Aspergillus oryzae-sourced protease and application thereof in bean fermentation products

Abstract

The invention relates to aspergillus oryzae sourced protease and application thereof in traditional bean fermentation products, and belongs to the technical field of enzyme engineering and microbial engineering. The amino acid sequence provided by the invention comprises an acid protease, two neutral proteases and an alkaline protease, wherein the proteases have higher degradation degree on globulin and albumin; the enzyme activity is the highest at 45-50 DEG C, and the salt tolerance is good. When the soybean sauce is prepared, the physicochemical indexes, free amino acids, organic acids and flavor substances of the soybean sauce are remarkably improved; wherein the quantity and the content of flavor substances are remarkably increased, and the content of free amino acid is increased by 20-25%; and the flavor sensory evaluation is more pleasant. In the early stage to the middle stage of fermentation, the content of amino acid nitrogen in the soy sauce is remarkably increased, the production cycle of the soy sauce can be shortened, and the method has wide application prospects in the food industry.

Description

Technical Field

[0001] The invention relates to a protease derived from Aspergillus oryzae and application thereof in traditional bean fermentation products, belonging to the technical fields of enzyme engineering and microbial engineering. Background Art

[0002] Aspergillus oryzae is an aerophilic fungus, belonging to the genus Aspergillus in taxonomy, and is a commonly used fermentation strain in industry. Aspergillus oryzae has the advantages of fast colony growth, many spores, and high enzyme activity. Aspergillus oryzae can secrete a variety of enzymes, including protease, amylase, cellulase, saccharifying enzyme, phytase, etc., so Aspergillus oryzae has strong protein decomposition and saccharification capabilities. At the same time, Aspergillus oryzae does not produce aflatoxin, grows rapidly, has low environmental requirements, can resist miscellaneous bacteria, has high enzyme activity, has no odor, and contributes to flavor.

[0003] Fermented bean products such as soy sauce and bean paste are produced by the traditional fermentation process of mold spores (koji) in a non-sterile environment. The production is divided into two stages, namely koji making and brine fermentation. By subculturing Aspergillus oryzae or Aspergillus sojae on grains (including steamed rice, wheat bran or soybean meal) or broad beans, and culturing in a warm environment (25-35°C), good ventilation and low humidity are maintained during the process to prevent the growth of undesirable microorganisms. Various enzymes are produced in mature koji, including proteases, α-amylases, cellulases and lipases. During the solid-state fermentation of koji, Aspergillus oryzae has leucine aminopeptidase and two endopeptidases (alkaline protease and neutral), while Aspergillus sojae also has acid carboxypeptidases, which hydrolyze soybean protein into polypeptides, peptides and amino acids. Starch and cellulose are enzymatically broken down into disaccharides and monosaccharides by extracellular α-amylase and cellulase, respectively. During the koji making process, the growth of undesirable microorganisms can be prevented by ensuring the growth of fungi, and enzymatic degradation processes can occur to provide substrates for microorganisms in subsequent fermentation. Placing the koji in salt water for fermentation can produce soy sauce or bean paste.

[0004] At present, Aspergillus oryzae Shanghai Brew 3.042 is widely used in my country as a fermentation agent for fermenting soy sauce and bean paste. The original strain was isolated from the soy sauce mash of Fujian Yongchun Soy Sauce Factory (China) by the Soy Sauce Industry Research Institute of the former Ministry of Food Industry in 1957. However, the strain has the disadvantages of being easy to change and having low acid protease production after continuous use.

[0005] Mining the information of Aspergillus oryzae protease can optimize the industrial production process, improve the quality and output of soy sauce industrial products, and reduce production costs, which is of great practical significance.

[0006] Aspergillus oryzae is one of the most important strains for producing proteases by microbial fermentation. It produces a wide variety of proteases, including acidic proteases, neutral proteases, alkaline proteases, etc. These proteases play a vital role in the fermentation process.

[0007] It is of great practical significance to study the information of Aspergillus oryzae protease, optimize the industrial production process, improve the quality and output of soy sauce industrial products, and reduce production costs. Summary of the invention

[0008] In order to solve the problems existing in the prior art, the present invention provides a key protease derived from Aspergillus oryzae and application thereof in bean fermentation products.

[0009] The first aspect of the present invention provides four Aspergillus oryzae-derived proteases, respectively named GME1689_g, GME238_g, GME3470_g and GME6033_g; the proteases are:

[0010] (a) a protein consisting of the amino acid sequence shown in SEQ ID No. 1 to SEQ ID No. 4; or,

[0011] (b) A protein derived from (a) in which the amino acid sequence in (a) is substituted, deleted or added with one or more amino acids and has protease activity.

[0012] The second aspect of the present invention provides a gene encoding the Aspergillus oryzae-derived protease, the gene sequence consisting of the nucleotides shown in SEQ ID No.5 to SEQ ID No.8.

[0013] The third aspect of the present invention provides a recombinant plasmid carrying the gene sequence.

[0014] Furthermore, the vector of the recombinant plasmid is a pPIC9k vector.

[0015] The fourth aspect of the present invention provides a host cell carrying the gene or the recombinant plasmid.

[0016] In some embodiments of the present invention, the host cell is Pichia pastoris.

[0017] The fifth aspect of the present invention provides a method for preparing the protease, wherein the host cells are added to a culture medium and cultured until the OD 600 =2.0-6.0, adding methanol to the fermentation broth at a certain interval to continue inducing the culture to obtain a fermentation broth containing the protease, and then separating and purifying to obtain the protease.

[0018] In some embodiments of the present invention, the culture medium comprises LB medium, YPD medium and BMMY medium.

[0019] In one embodiment of the present invention, methanol is added to the fermentation broth every 24 hours and the induction culture is continued for 4 days to obtain the protease.

[0020] In a preferred embodiment, the amount of methanol added to the fermentation broth is 1%, and it is added once every 24 hours.

[0021] In one embodiment of the present invention, the culture temperature is 30° C. and the rotation speed is 200 rpm.

[0022] The sixth aspect of the present invention provides the Aspergillus oryzae-derived protease, the gene, the recombinant plasmid, the host cell, or use in bean fermentation products.

[0023] In some embodiments of the present invention, the bean fermentation product includes but is not limited to fermented black beans, bean paste, and soy sauce; further, the bean paste includes but is not limited to broad bean paste and soybean paste.

[0024] In some embodiments of the present invention, the Aspergillus oryzae-derived protease is used to ferment and prepare soy sauce; the protease is added at the initial stage of soy sauce fermentation to obtain soy sauce with improved quality.

[0025] Furthermore, when the soy sauce is fermented into koji and mixed with salt water to start fermentation, the Aspergillus oryzae-derived protease is added in an amount of 100-1000 U / kg;

[0026] In a preferred embodiment, the added amount of protease is 200 U / kg.

[0027] In some embodiments of the present invention, the salinity of the sauce mash is 80-180 g / L;

[0028] In some embodiments of the present invention, the soy sauce fermentation temperature is 25-40°C;

[0029] In some embodiments of the present invention, the soy sauce is fermented for a total of 10-12 weeks.

[0030] In one embodiment of the present invention, the method for preparing soy sauce comprises the steps of:

[0031] S1. Koji making: Mix soybean meal and bran in a ratio of 3:2, moisten with 100% water, cook at 121℃ for 20min, cool to below 40℃, and heat for 10min. 7 / g spore inoculation amount was inoculated into the material with Aspergillus oryzae BL18 strain, and the temperature was controlled at 30°C and the humidity was controlled at 95% to culture for 72h to obtain finished koji;

[0032] S2. Fermentation: adding salt water with a salt concentration of 12-27% to the finished koji in a ratio of 1:2, adding the Aspergillus oryzae-derived protease, and then putting it into a fermentation tank for fermentation. After the fermentation is completed, filtering the mash.

[0033] Further, the Aspergillus oryzae-derived protease is selected from one or a combination of GME1689_g, GME238_g, GME3470_g, and GME6033_g;

[0034] More preferably, the Aspergillus oryzae-derived protease is GME1689_g, GME238_g or GME1689_g+GME238_g.

[0035] In some embodiments of the present invention, the mass ratio of GME1689_g+GME238_g is 1:1.

[0036] Beneficial Effects

[0037] (1) The present invention first provides a protease derived from Aspergillus oryzae, comprising an acidic protease, two neutral proteases and an alkaline protease, the amino acid sequences of which are shown in SEQ ID No. 1 to SEQ ID No. 4; the protease has a high degree of degradation for globulin and albumin; has good enzyme activity at 45-50° C. and has good salt tolerance;

[0038] (2) The present invention also provides a gene encoding the protease, a recombinant plasmid carrying the gene, a vector of the plasmid, a host cell, and a method for preparing the protease;

[0039] (3) The Aspergillus oryzae-derived protease provided by the present invention has broad application prospects in food fermentation. When used to prepare soy sauce, the physicochemical indicators, free amino acids, organic acids and flavor substances of the soy sauce are significantly improved. Among them, the quantity and content of flavor substances are significantly improved, and the free amino acid content is increased by 20-25%. The flavor sensory evaluation is also more pleasant.

[0040] (4) In the early to middle stages of fermentation, the addition of protease significantly increased the content of amino acid nitrogen in soy sauce and shortened the soy sauce production cycle. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] The accompanying drawings are used to provide a further understanding of the present application and constitute a part of the specification. Together with the embodiments of the present application, they are used to explain the present application and do not constitute a limitation of the present application. In the accompanying drawings:

[0042] Figure 1 :The expression curve of secretable protease gene of Aspergillus oryzae BL18 during koji making process;

[0043] Figure 2 : Reconstructed recombinant plasmid map;

[0044] Figure 3: SDS-PAGE gel images of recombinant proteases GME1689_g (1), GME238_g (2), GME3470_g (3) and GME6033_g (4) after expression and purification;

[0045] Figure 4 :Changes in the activity of recombinant protease under different temperature conditions;

[0046] Figure 5 : Optimal pH line graph of the recombinant protease (A) and residual enzyme activity after storage at different pH for 24 h (B);

[0047] Figure 6 : Changes in the enzymatic activity of the recombinant protease at 10% salinity (A), 14% salinity (B) and 18% salinity (C);

[0048] Figure 7 :Degradation rate curves of globulin (A) and albumin (B) by recombinant protease;

[0049] Figure 8 : Analysis of degradation products of globulin (A) and albumin (B) by recombinant proteases;

[0050] Fig. 9 :Effects of recombinant protease addition on pH (A), amino acid nitrogen (B), total acid (C) and reducing sugar (D) during soy sauce fermentation;

[0051] Fig.10 :The effect of recombinant protease addition on free amino acids (A) and organic acids (B) in soy sauce fermentation samples;

[0052] Fig.11 :The effect of adding recombinant protease on the quantity (A) and content (B) of volatile flavor substances in soy sauce fermentation samples;

[0053] Fig.12 : Effects of recombinant protease addition on the sensory properties of soy sauce fermentation samples. DETAILED DESCRIPTION

[0054] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the embodiments of the specification.

[0055] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein, and those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0056] Aspergillus oryzae BL18 involved in the following examples was deposited by the General Microbiology Center of the China Culture Collection Administration on May 6, 2020, with a deposit number of CMCC No. 19628, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

[0057] An invention patent has been authorized (the patent name is a strain of Aspergillus oryzae producing high protease activity and its application in soybean paste fermentation, patent number is ZL202010661413.X); the Escherichia coli (E. coli JM109), Pichia pastoris (Komagataellaphaffii GS115) and pPIC9k involved in the following embodiments are all preserved in the Brewing Science and Engineering Research Laboratory, School of Bioengineering, Jiangnan University.

[0058] The culture medium involved in the following examples is as follows:

[0059] PDA solid culture medium: purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0060] LB liquid medium: peptone 10 g L -1 , yeast extract 5g·L -1 , sodium chloride 10g·L -1 , ampicillin 100 μg mL -1 ;

[0061] LB solid medium: peptone 10 g L -1 , yeast extract 5g·L -1 , sodium chloride 10g·L -1 , agar 20 g·L -1 , ampicillin 100 μg mL -1 ;

[0062] YPD liquid medium: yeast extract 10 g L -1 , peptone 20 g·L -1 , glucose 20g·L -1 , ampicillin 100 μg mL -1 ;

[0063] YPD solid medium: yeast extract 10 g L -1 , peptone 20 g·L -1 , glucose 20g·L -1 , agar 20 g·L -1 , ampicillin 100 μg mL -1 ;

[0064] BMGY liquid medium: peptone 20.0 g·L -1, yeast extract powder 10.0g·L -1 , phosphate buffer (pH 6.0) 100 mmol·mL -1 , YNB (amino-free yeast nitrogen source) 13.4 g·L -1 , glycerol: 10.0 g·L -1 , biotin 100 μg mL -1 , ampicillin 100 μg mL -1 ;

[0065] Extraction of globulin and albumin from broad beans: weigh 5g broad beans, add 50mL of distilled water, shake and extract at 40℃ for 1h, centrifuge at 6000rpm, rinse the precipitate three times with distilled water, combine the supernatant with the first after centrifugation, and make the volume to 100mL, which is the albumin solution (water-soluble protein); add 50mL of 5% NaCl solution to the precipitate, shake and extract at 40℃ for 1h, centrifuge at 6000rpm, rinse the precipitate three times with 5% NaCl solution, combine the supernatant with the first after centrifugation, and make the volume to 100mL, which is the globulin solution (salt-soluble protein).

[0066] Example 1: Analysis of key proteases of Aspergillus oryzae BL18 strain

[0067] The specific steps are as follows:

[0068] Whole-genome sequencing of Aspergillus oryzae BL18 and 3.042: Genomic DNA of Aspergillus oryzae BL18 was extracted using a magnetic plant genomic DNA kit (BGI, Shenzhen, China). After confirming the concentration, purity, and integrity of the extracted DNA, whole-genome sequencing was performed on the PacBio sequel system with the help of BGI (Shenzhen, China). The concentration of the DNA samples was detected by Qubit fluorescence quantification, and the integrity of the DNA samples was detected by 1% agarose gel electrophoresis. Only the samples that passed the test were used for library preparation. The DNA samples were fragmented by ultrasound using a Covaris instrument, and the short DNA fragments of the desired length were obtained by adjusting the fragmentation parameters. The fragmented samples were fragmented using AgencourtAMPure XP Medium. The samples were selected by the AgencourtAMPure XP Medium kit to ensure that the sample bands were concentrated around 300-400 bp; the amount of the purified DNA samples was detected using the Qubit dsDNAHS detection kit 500. The reaction system was prepared and reacted at an appropriate temperature for a period of time to repair the ends of the double-stranded DNA and add a base to the 3' end. Prepare the ligation reaction system and react at an appropriate temperature for a period of time to connect the connection point to the DNA. Prepare the PCR reaction system and set the reaction program to amplify the connection product. The amplified product was screened for fragments using AgencourtAMPure XP medium, and the PCR product was detected using an Agilent 2100 bioanalyzer. The PCR product was denatured into a single strand, and then the circularization reaction system was prepared. The single-stranded circular product was obtained by sufficient mixing and reacting at an appropriate temperature for a period of time, and the final library was obtained after digesting the uncircularized linear DNA molecules. The Agilent 2100 bioanalyzer was used to detect the fragment size and concentration of the library. The single-stranded circular DNA molecules replicated by rolling circles to form DNA nanoballs (DNBs) containing more than 300 copies. The resulting DNBs were incorporated into the mesh holes on the chip using high-density DNA nanochip technology. Sequencing was performed by co-probe anchored polymerization (cPAS). Library construction was performed using the PacBio platform. First, 1 μg of genomic DNA was sheared into 10-15 kb fragments using a g-TUBE device. Then, the DNA was sequenced using a DNA nanoarray. Express template preparation kit 2.0 was used to construct the library, and finally the SMRTcell library was sequenced using the sequel (PacBio) sequencing kit 2.0.

[0069] Mining of genomic data: After obtaining the raw sequencing data, the raw data were first filtered to find reads containing sequencing adapters, low-quality base ratios, and highly unknown base ratios. The filtered high-quality data were subjected to statistical analysis and downstream bioinformatics analysis. The genome was assembled by subad correction, correction read assembly, assembly result correction, scaffold construction, and gap filling. The software used for de novo annotation included Genewise, SNAP, Augustus, and Genemarkes. The genome information of Aspergillus oryzae 3.042 (Genbank WGS accession number AKHY01) was used as a reference. Functional annotation was performed using multiple databases, including the non-redundant protein sequence database (NR), the JGI Encyclopedia of Genomes and Genomes (KEGG), the protein homology group cluster (COG), the gene ontology (GO) database, the carbohydrate-active enzyme (CAZy) database, EggNOG, and the Swiss-Prot database, following the default parameters. The raw data and genome assembly results are available at NCBI (accession number JAJRD00000000 and Bioproject number PRJNA776566).

[0070] Prediction and analysis of hydrolase genes in the Aspergillus oryzae BL18 genome: The gene sets obtained from the sequencing of Aspergillus oryzae 3.042 and BL18 strains were annotated in 16 databases, including P450, CAZY, KINASE, SWISSPROT, NOG, COG, CARD, NR, DBCAN, TCDB, IPR, PHI, KEGG, GO, phosphatase, and KOG. Genes with hydrolase function were screened out by comparing parameters with functional annotations such as Identity, E_value, and Score. Signal peptides of hydrolases were predicted using SignalP4.0 software using a neural network approach and a hidden Markov model. Transmembrane proteins were analyzed by TMHMM v2.0 software to identify potential hydrolases with potential transmembrane domains. Potential hydrolases that can be secreted outside the cell are defined as enzymes with signal peptides and lack a transmembrane domain.

[0071] Based on the analysis of the Aspergillus oryzae genome, 38 secretory protease genes were identified. On this basis, the gene expression of the above 38 secretory protease genes during the koji making process of fermented bean foods was further analyzed.

[0072] like Figure 1As shown, the expression of 38 secretory protease genes in Aspergillus oryzae BL18 and Aspergillus oryzae 3.042 samples all showed a downward trend during the 24h, 48h, and 72h of koji making. When koji making 24h, the expression of protease genes was at a higher level, and most of the protease gene expression levels decreased sharply afterwards, which shows that the early stage of koji making is an important period for Aspergillus oryzae BL18 to produce protease. Among 38 Aspergillus oryzae protease genes, endoprotease gene GME238_g (encoding Alkaline protease2) and GME6033_g (encoding Vacuolar protease A) and exoprotease gene GME1689_g (encoding tripeptidyl-peptidase I) and GME3470_g (encoding Carboxypeptidase Y) have higher expression in the early stage of Aspergillus oryzae BL18 koji making, which may be the key protease gene of Aspergillus oryzae in the koji making process.

[0073] Example 2: Heterologous expression of protease genes in Pichia pastoris

[0074] The specific steps are as follows:

[0075] 1. Construction and transformation of recombinant plasmids containing 4 highly expressed protease genes

[0076] The cDNA library of Aspergillus oryzae was obtained by extracting RNA from Aspergillus oryzae BL18 and then reverse transcription. In order to obtain RNA from Aspergillus oryzae BL18, Aspergillus oryzae BL18 was inoculated into PDA solid medium and cultured at 30°C for 72 hours, at which time the mycelium was green. The RNA of Aspergillus oryzae BL18 was extracted using a column-type fungal total RNA extraction and purification kit. Ⅲ1st StrandcDNA Synthesis SuperMix synthesizes the first strand cDNA. The reverse transcription reaction mixture contains ⅢSuperMix5μL, total RNA 1μL, RNase-free H2O 14μL; reverse transcription program is 25℃5min, 55℃15min, 85℃5min. The cDNA was obtained by PCR using the first chain as a template. The PCR mixture contained 25μL 2×PhantaMax Master Mix (DyePlus), 1.5μL primer 1 and primer 2, the above reverse transcription product as a template 2μL, ddH2O 20μL. The PCR program was 95℃ pre-denaturation for 3min and then 35 cycles of 95℃ denaturation for 15s, 58℃ annealing for 15s, 72℃ extension for 120s, and finally repeated at 72℃ for 5min to extend completely. The above-obtained gene was connected to the pPIC9k plasmid by homologous recombination technology and transformed into Escherichia coli JM109 for plasmid amplification, and the recombinant plasmid was verified by double enzyme digestion ( Figure 2 ). The correct recombinant plasmid was further transformed into Pichia pastoris GS115 by electroporation.

[0077] Table 1 Primer sequences

[0078]

[0079]

[0080] 2. Expression and purification of protease

[0081] The recombinant bacteria K.phaffii GS115 / pPIC9K-GME1689_g, K.phaffii GS115 / pPIC9K-GME238_g, K.phaffii GS115 / pPIC9K-GME3470_g and K.phaffii obtained in Example 1 were selected and purified. The GS115 / pPIC9K-GME6033_g recombinant strain was spread on YPD solid medium, cultured at 30°C for 3-4 days, and the grown single colonies were picked and inoculated into YPD liquid medium containing 100 μg / mL ampicillin, cultured at 30°C and 220 rpm for 1 day, and transferred to BMGY liquid medium containing 100 μg / mL ampicillin at an inoculum volume of 4% (v / v), cultured at 30°C and 220 rpm for 1 day, 1% methanol was added, and the expression was continued at 30°C and 220 rpm for 4 days to obtain a fermentation broth; the fermentation broth was heated at 5000 rpm for 24 hours. Centrifuge for 20 minutes, keep the supernatant, collect the cells and resuspend them in 50mM phosphate buffer (pH6.5) to obtain a cell resuspension; ultrasonically disrupt the cell resuspension in an ice bath, the disruption conditions are: 20% power ultrasound for 4s, interval 4s, 120 times, to obtain a cell disruption solution; centrifuge the cell disruption solution at 4°C, 8000rpm for 20 minutes to obtain a cell disruption supernatant, which is mixed with the above supernatant; the mixed solution is subjected to nickel column affinity chromatography to obtain purified GME1689_g, GME238_g, GME3470_g and GME6033_g. SDS-PAGE analysis found that the purified GME1689_g, GME238_g, GME3470_g and GME6033_g had single bands, and their sizes were consistent with their theoretical molecular weights ( Figure 3 ).

[0082] Example 3: Analysis of enzymatic properties of four proteases

[0083] The specific steps are as follows:

[0084] Accurately weigh 0.1000 g ± 0.0002 g of L-tyrosine that has been dried to constant weight at 105 °C, dissolve it in 60 mL of 1 M hydrochloric acid solution and adjust the volume to 100 mL, which is 1 mg mL -1 Tyrosine solution. Pipette 1 mg·mL -1 10.00 mL of tyrosine solution was diluted to 100 mL with 0.1 M hydrochloric acid solution to obtain 100 μg mL -1 L-tyrosine standard stock solution was prepared. L-tyrosine solution was diluted to 0, 10, 20, 30, 40 and 50 μg mL -1 , measure the OD of fructose solution with different concentrations 600, draw a standard curve with the absorbance value as the ordinate and the corresponding L-tyrosine concentration as the abscissa;

[0085] The protease activity was determined by the Folin phenol reagent method. Casein solutions (10.0 g·L) at pH 3.0, pH 7.2, and pH 10.0 were prepared respectively. -1 ), 1 mL of the purified sample was incubated with the casein solution for 10 min, and 2 mL of trichloroacetic acid was added to terminate the reaction. The precipitate was removed by filtration or centrifugation, and the supernatant was taken and Na2CO3 (20 g·L -1 )5mL and diluted Folin phenol (42.4g·L -1 )1mL of reaction solution was mixed and kept at 40℃ for 20min to complete the reaction. The absorbance wavelength was 680nm. Each sample should be repeated 3 times and a blank control should be made at the same time.

[0086] One unit of protease activity is defined as the amount of enzyme required to decompose casein and produce 1 μg of tyrosine per minute at 40°C;

[0087] The specific activity of protease is defined as the number of enzyme activity units (U) per unit weight (mg) of protease at pH 7.2 and 40°C.

[0088] 1. Optimum reaction temperature

[0089] The activity of the purified protease obtained in Example 2 was measured at reaction temperatures of 20, 30, 35, 40, 45, 50, 55, 60, 65, 70 and 80°C, respectively. The highest activity was taken as 100%, and the relative activities of the remaining activities were calculated compared with the temperature to investigate the optimal reaction temperature of the enzyme.

[0090] Depend on Figure 4 It can be seen that the enzymatic activities of the four proteases gradually increased between 20°C and 50°C, but decreased rapidly when the temperature exceeded 50°C. The optimum temperature of GME238_g and GME3470_g enzymes was 45°C, while the optimum temperature of GME1689_g and GME6033_g enzymes was 50°C.

[0091] 2. Optimal reaction pH

[0092] Casein solutions with pH values ​​of 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, and 12.0 were prepared, and the activity of the purified protease obtained in Example 2 was measured at the corresponding optimal temperature. The highest activity was taken as 100%, and the remaining activities were compared with it to calculate the relative activity, so as to investigate the optimal pH of the enzyme. Figure 5A shows that the GME1689_g protease has enzymatic activity in the pH range of 4-11, with the highest enzyme activity at pH 7. The optimum pH value of the GME238_g protease is pH 8, and it has enzymatic activity at pH 5-9. The optimum pH value of the GME3470_g protease is pH 5, and it has greater than 60% enzymatic activity at pH 5-8. The optimum pH value of the GME6033_g protease is pH 6, and it has greater than 60% enzymatic activity at pH 5-7.

[0093] 3. pH stability determination

[0094] Casein solutions with pH values ​​of 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, and 12.0 were prepared, and the purified protease obtained in Example 2 was stored at 25°C for 1 hour in the above buffer system, and then its enzyme activity was measured at different temperatures, with the highest enzyme activity being 100%, and the remaining enzyme activities were compared with it to calculate the relative enzyme activity to examine its pH stability. Figure 5 B shows that after 1h of treatment at pH 3-12, GME1689_g has almost no enzyme activity loss at pH 7, only about 20% enzyme activity at pH 6 and pH 8, and almost all enzyme activity is lost under other pH conditions; GME238_g has a residual enzyme activity close to 100% at pH 8, and more than 60% at pH 7, and more than 20% at pH 5-6 and pH 9; GME3470_g has a relatively high residual enzyme activity at pH 4-7. When the pH exceeds 7, the enzyme is rapidly inactivated; after 1h of treatment at pH 4 and pH 5, GME6033_g still has more than 60% residual enzyme activity, and when the pH value is increased to more than pH 6, the residual enzyme activity is significantly reduced.

[0095] 4. Effects of different salinities on proteolytic enzymes

[0096] The proteolytic enzyme solution (200 U / mL) was mixed with the NaCl solution to obtain a mixture of different salinities (10%, 14%, 18%), and samples were collected every 24 hours. The protease activity of the collected samples was measured and an inactivation curve was drawn. The proteolytic enzyme activity measured at the beginning of the treatment was recorded as 100%, and the other enzyme activities were expressed as relative percentages of enzyme activity. Figure 6 As shown, GME1689_g protease has the best salt tolerance, GME238_g protease is second, and GME6033_g protease has the worst salt tolerance.

[0097] Example 4: Analysis of kinetic parameters of protease

[0098] The specific steps are as follows:

[0099] In the study of enzyme kinetics, Km and K cat It is the most important characteristic constant of the enzyme-catalyzed reaction, which reflects the affinity between the enzyme and the substrate and the reaction rate of the enzyme. -1 200 μL of casein as substrate, 1000-fold diluted purified protease obtained in Example 2 was added, and the reaction was carried out at the corresponding optimal temperature and optimal pH conditions for 10 min, and the amount of tyrosine generated per unit time was taken as the initial velocity V of the enzyme reaction; according to the Michaelis-Menten equation V = {Vmax · [S]} / {Km + [S]}, the initial velocity V of the enzyme reaction and the substrate concentration [S] were nonlinearly fitted to calculate the Km value and Vmax value of the protease, and the Kcat value and K cat / K m value.

[0100] As shown in Table 2, the specific activities of GME1689_g, GME238_g, GME3470_g and GME6033_g were 2218.93 U / mg, 2455.53 U / mg, 961.11 U / mg and 1654.20 U / mg, respectively, and their Michaelis constants were 0.33 mM, 0.30 mM, 0.31 mM and 0.27 mM, respectively. After calculation, the k cat / K m The values ​​are 7.70min -1 mM -1 、5.23min -1 mM -1 、5.58min -1 mM -1 and 12.26min -1 mM -1 .

[0101] Table 2 Enzyme activities, specific enzyme activities and enzyme kinetic parameters of the four enzymes after purification

[0102]

[0103] Example 5: Analysis of the ability of proteases to degrade globulin and albumin

[0104] The specific steps are as follows:

[0105] The ratio of enzyme to substrate was 100:1 (U / g) for the determination of the degree of protease hydrolysis, and the reaction time was 120 min. At 50°C and pH 7, samples were taken at 0, 15, 30, 45, 60 and 120 min, and immediately heated at 90°C for 10 min to stop the enzyme reaction. The total amount of hydrolysis was determined by trinitrobenzene sulfonic acid (TNBS) reaction. The same reaction time at 50°C and pH 7 was used as a control to eliminate the effects of temperature and pH on the degree of hydrolysis; globulin and albumin were hydrolyzed with 6-N-HCl at 110°C to determine the total number of amino groups in the sample.

[0106] like Figure 7 As shown in the figure, the degradation rate of globulin and albumin by the four proteolytic enzymes was relatively fast at the beginning of the treatment, and the degradation rate gradually slowed down after 30-40 hours. Figure 7 As shown in A, GME1689_g and GME238_g have higher degradation capacity for albumin and globulin than the other two proteolytic enzymes. After 120 minutes of treatment, the degradation degree of albumin by the four enzymes was GME1689_g (29.89%), GME238_g (28.17%), GME3470_g (25.84%) and GME6033_g (24.32%). Figure 7 As shown in B, after 120 minutes of hydrolysis, the degradation degrees of globulin by the four enzymes were GME1689_g (30.84%), GME238_g (35.22%), GME3470_g (28.55%), and GME6033_g (27.47%), respectively.

[0107] The two proteolytic enzymes (GME1689_g and GME238_g) with the highest hydrolysis degree of globulin and albumin were added in equal amounts to the albumin or globulin solution. The hydrolysis degree values ​​of globulin and albumin increased faster in the group with the two proteases. After 120 minutes of treatment, the degradation degree of albumin and globulin by the composite enzyme was 49.91% and 56.31%, respectively, which was significantly higher than the degradation degree of the two enzymes used alone (p<0.05). After 30 minutes of treatment, the degradation degree of albumin or globulin by the composite enzyme was similar to that of the enzyme used alone after 120 minutes of treatment.

[0108] Gel filtration chromatography (GFC) was used to determine the polypeptide content to determine the degree of enzymatic hydrolysis. The enzyme and substrate were 100:1 (U / g), reacted at 50°C for 60 minutes, and immediately heated at 90°C for 10 minutes to stop the enzyme reaction. 100 μL of the sample was diluted to 10 mL with water, ultrasonicated in an ultrasonic cleaner at room temperature for 5 minutes, centrifuged, and 400 μL of the supernatant was filtered through a 0.22 μm aqueous phase filter membrane, and then measured using a Waters 2695 high performance liquid chromatograph (equipped with a 2487 UV detector and Empower workstation GPC software). Liquid phase conditions: chromatographic column: TSKgel 2000SWXL 300mm×7.8mm, column temperature: 30℃, detection: UV220 nm, flow rate: 0.5mL / min, standards used for molecular weight calibration curve: cytochrome C (MW12384), aprotinin (MW6500), bacitracin (MW1422), tyrosine-tyrosine-tyrosine-arginine (MW451) and tyrosine-tyrosine-tyrosine (MW189).

[0109] like Figure 8 As shown in A, the four enzymes can degrade the large molecular polypeptides (MV>10000) of albumin (5C) and globulin (5D) into small molecular polypeptides (MV<500). Before enzymatic hydrolysis, the contents of large molecular polypeptides and small molecular polypeptides in the albumin solution were 62.36% and 25.99%, respectively. After enzymatic hydrolysis by GME1689_g, GME238_g, GME3470_g, GME6033_g and the composite protease of GME1689_g and GME238_g, the contents of large molecular polypeptides in the solution were reduced to 5.34%, 20.11%, 16.36%, 19.18%, and 10.24%, respectively, while the contents of small molecular polypeptides were increased to 87.02%, 75.9%, 74.52%, 72.16%, and 84.46%, respectively. Figure 8 A). It can be seen that GME1689_g protease has the highest degree of hydrolysis of large molecular polypeptides in albumin, followed by GME238_g. When these two enzymes are added at the same time, the hydrolysis degree of small molecular polypeptides is similar to that of GME1689_g protease alone.

[0110] like Figure 8As shown in Figure B, the content of macromolecular polypeptides in the globulin solution after enzymatic hydrolysis by GME1689_g, GME238_g, GME3470_g, GME6033_g and GME1689_g+GME238_g proteases was 6.35%, 1.73%, 8.75% and 8.34%, 1.47%, respectively, which was significantly lower than the content of macromolecular polypeptides in the globulin solution (35.4%); while the content of small molecular polypeptides in the solution after being mediated by the four proteases was 90.68%, 97.74%, 88.42%, 88.84%, 88.14%, respectively, which was significantly higher than the corresponding content before enzymatic hydrolysis (53.42%). Among them, GME238_g protease had the highest degree of hydrolysis of macromolecular polypeptides in the globulin solution, followed by GME1689_g, but after hydrolysis by GME1689_g protease, the content of small molecular polypeptides with MV<180 in the solution was higher, and the polypeptides could be hydrolyzed more thoroughly. When GME1689_g and GME238_g were added at the same time, the content of large molecular weight polypeptides was the lowest, and the content of small molecular weight polypeptides was also the highest. The above results were consistent with the hydrolysis rate results.

[0111] GME1689_g and GME238_g showed higher degradation capacity for albumin and globulin than the other two proteases. The hydrolysis degree of GME1689_g and GME238_g was significantly higher than that of single enzymes.

[0112] Example 6: Effect of protease on soy sauce fermentation

[0113] 1. Koji making: Mix soybean meal and bran in a ratio of 3:2, moisten with 100% water, sterilize at 121°C for 20 minutes, cool to below 40°C, and add 10 7 / g of spores were inoculated into Aspergillus oryzae BL18 strain, and the temperature was controlled at 30°C and the humidity was 95% and cultured for 72h.

[0114] 2. Fermentation: Add salt water with a salt concentration of 12% to the finished koji at a ratio of 1:2, and after mixing, divide the mixture into four equal parts, add purified protease GME1689_g, GME238_g, GME1689_g+GME238_g (mass ratio is 1:1) and an equal volume of deionized water, and use the group without enzyme as the control group. The enzyme addition amount is 200U / kg, and the fermentation is carried out in a barrel for 80 days.

[0115] Determination of physicochemical parameters of soy sauce: After fully stirring the soy sauce, accurately weigh 5g of the sample and dilute it to 100mL with deionized water. After centrifugation at 8000rpm, collect the supernatant and filter it with double-layer filter paper. The pH value of the collected sample is measured by a pH meter. The titratable acid concentration is determined by acid-base titration. The reducing sugar content in soy sauce is determined by direct titration. The acidometer method is used to determine amino acid nitrogen. Each experiment is repeated three times.

[0116] like Fig. 9 As shown in A, the pH value of soy sauce in all groups decreased rapidly in the early stage of fermentation (0-30 days) and stabilized in the middle and late stages of fermentation (30-80 days). The addition of protease can promote the accumulation of acidic substances in soy sauce, thereby accelerating the decline in pH value. Among them, the pH value of the soy sauce sample with the addition of GME1689_g protease decreased the fastest, and its pH value dropped to about 5.2 after fermentation. The pH value of the soy sauce with the addition of GME238_g and a mixture of the two enzymes was around 5.5, slightly lower than the control group (pH5.6).

[0117] like Fig. 9 As shown in Figure C, the effect of protease on total acid accumulation in soy sauce is consistent with the change in pH. The addition of GME1689_g protease is conducive to the generation of acidic substances, resulting in an increase in total acid concentration after fermentation. After fermentation, the total acid concentrations in soy sauce samples prepared with GME1689_g, GME238_g, and composite enzymes were 3.63 g / 100 g, 3.56 g / 100 g, and 3.61 g / 100 g, respectively, all higher than the control group (3.39 g / 100 g, p<0.05).

[0118] like Fig. 9 As shown in B, amino acid nitrogen is a key indicator of soy sauce quality and is often used as a standard for soy sauce grading. Protein hydrolysis products, such as amino acids and oligopeptides, are the main sources of amino acid nitrogen. The amino acid nitrogen in soy sauce increases rapidly in the early stage (0-30 days) and tends to reach a peak in the middle and late stages (30-80 days). In the early to middle stages of fermentation, the addition of protease significantly increased the content of amino acid nitrogen in soy sauce. After fermentation was completed, the concentrations of amino acid nitrogen in the soy sauce samples added with GME1689_g, GME238_g and both enzymes were 0.54g / 100g, 0.52g / 100g and 0.53g / 100g, respectively.

[0119] Analysis of free amino acids in soy sauce: 1.0 g of soy sauce sample was collected at the end of fermentation and dissolved in 5% (w / v) trichloroacetic acid solution to 25 mL. The obtained solution was ultrasonically treated at 25°C for 20 minutes and allowed to stand for 2 hours. After filtering through a double layer of filter paper, 1 mL of the clarified filtrate was transferred to a 1.5 mL centrifuge tube and centrifuged at 12000 rpm for 10 minutes. 400 μL of the supernatant was filtered through a 0.45 μm aqueous filter membrane, and the free amino acid concentration was analyzed by an Agilent 100 HPLC equipped with an ODS column (250 mm × 4.6 mm, 5 μm). The injection volume was 10 μL; the column temperature was 40°C; the UV detection wavelength was 338 nm (proline 262 nm); the mobile phase A was sodium acetate solution (20 mM), and the mobile phase B was sodium acetate solution: methanol: acetonitrile (1:2:2, v / v); the elution method was isocratic elution, and the flow rate was 1.0 mL / min.

[0120] like Fig.10 As shown in A, the addition of protease significantly increased the concentration of free amino acids in soy sauce. The total amino acid concentrations in the soy sauce samples with GME1689_g or GME238_g added alone and both added together were 97.82 mg / 100 mL, 94.79 mg / 100 mL, and 96.24 mg / 100 mL, respectively, which were 24.72%, 20.86%, and 22.71% higher than the control group (78.43 mg / 100 mL), respectively.

[0121] Determination of organic acids in soy sauce: Preparation of standard curve: Accurately weigh 0.1g of each common organic acid in soy sauce (oxalic acid standard, formic acid, malic acid, lactic acid, acetic acid, citric acid and succinic acid), dilute to 100mL with mobile phase, and prepare standard stock solutions. Dilute each organic acid standard stock solution to different concentrations (oxalic acid: 1, 10, 25, 50, 75 and 100mg / L; malic acid, lactic acid, acetic acid, citric acid and succinic acid: 10, 20, 250, 500, 750 and 1000mg / L). Analyze the concentration of the standard solution by HPLC and draw a standard curve. Accurately weigh 5.0g of ground soy sauce sample and heat to a slight boil. After cooling, add 0.5mL of 15% (w / v) potassium ferrocyanide solution and 0.5mL of 30% (w / v) zinc sulfate solution in sequence, and fill to 50mL with mobile phase. After standing for 30 minutes, the solution was filtered through filter paper and 0.45 μm organic filter membrane, and then the organic acid content was determined by HPLC equipped with Waters Atlantis T3 column (250 mm × 4.6 mm, 5 μm). The injection volume was 10 μL; the column temperature was 55 ° C; the UV detection wavelength was 210 nm; the mobile phase was H2O:2.5mM H2SO4 (3:1, v / v, pH 2.7); the flow rate of isocratic elution was 0.6 mL / min.

[0122] like Fig.10 As shown in Figure B, the addition of GME1689_g and GME238_g proteases alone or together significantly increased the concentration of organic acids, especially acetic acid and succinic acid. The concentrations of oxalic acid (2.45 mg / 100 g), formic acid (7.96 mg / 100 g), malic acid (13.64 mg / 100 g), and succinic acid (1015.34 mg / 100 g) were the highest in the soy sauce sample treated with GME238_g protease, but the concentration of lactic acid (15.91 mg / 100 g) was the lowest. When the two enzymes were added together, the total acid concentration (1025.66 mg / 100 g) was significantly lower than that of GME238_g protease (1142.91 mg / 100 g, p<0.05).

[0123] Analysis of volatile flavor compounds (VFCs) in soy sauce: Samples were collected after 80 days of fermentation and analyzed for volatile flavor substances by headspace solid phase microextraction gas chromatography-mass spectrometry (HS-SPME GC-MS). Soy sauce samples (2.0 g) were mixed with 2.0 g NaCl and 6 mL ddH2O in a 15 mL gas phase bottle, and 5 μL 2-octanol (102.8 mg / L) was added as an internal standard. Volatile flavor substances were analyzed using the HS-SPME GC-MS method equipped with an OV-1701 capillary column (30 mm×0.25 mm, 0.25 μm). The sample inlet temperature was 250°C, and the GC column temperature started at 32°C, increased to 180°C at a rate of 5°C min, and then increased to 240°C at 12°C / min and maintained for 5 minutes. The carrier gas (He) was used at a flow rate of 0.8 mL / min without pyrolysis. GC-MS chromatograms of volatile flavor compounds were obtained using an EI ionization source at 70 eV and matched to the NSIT and Willey spectral libraries. Only identifiers with a match value greater than 700 were used. 2-Octanol was used as an internal standard to quantify the relative amount of each flavor compound in the sample.

[0124] like Fig.11 As shown in the figure, 119 flavor substances were identified in the control group, while the corresponding values ​​were 140, 128 and 129 in the samples treated with GME1689_g, GME238_g alone or both. This indicates that the addition of enzymes enhances the production of flavor substances during soy sauce fermentation, among which the number of flavor substances is mainly enriched in alcohols, acids and alkanes. The concentration of flavor substances in soy sauce samples treated with GME238_g (1642.20μg / kg) and treated with both enzymes (2192.59μg / kg) was also significantly higher than that in the control group (1152.61μg / kg, p<0.05).

[0125] Sensory evaluation: The sensory evaluation of soy sauce samples was conducted according to the rules described in Section 3 of the Chinese national standard GB / T 5009.39-1996, including color, appearance, aroma, taste and typicality. A panel of 20 trained sensory assessors (10 males and 10 females) was invited to evaluate the flavor characteristics of the soy sauce samples. The sensory evaluation was conducted at 20-25°C with a double-blind experimental design. The 10-degree method was used, in which 10 points and 0 points represent the best or worst intensity. Fig.12 As shown in the figure, among the enzyme treatment groups, the soy sauce treated with GME1689_g scored higher in color, aroma and taste than the other two groups, while the three groups scored similarly in appearance and typicality. Compared with the control group, the sensory characteristics of the soy sauce with added protease showed brighter and relatively darker color, stronger sauce aroma and thick taste. This shows that the application of these two proteases, especially GME1689_g, improves the quality of soy sauce.

[0126] The addition of protease will promote the accumulation of acidic substances in soy sauce, thereby accelerating the decrease of pH value and significantly increasing the total acid in soy sauce; and significantly increase the amino acid nitrogen content of soy sauce in the early and middle stages of fermentation, but gradually tend to be consistent in the middle and late stages of fermentation; after adding protease, the reducing sugar content in soy sauce is lower than that in the control group ( Fig. 9 D). The addition of protease also increases the content of free amino acids and organic acids, which can give soy sauce a more pleasant taste ( Fig.10 ), indicating that the addition of enzymes promoted the formation of flavor substances during soy sauce fermentation, among which the amount of flavor substances was mainly enriched in alcohols, acids and alkanes ( Fig.11 In the sensory evaluation, the enzyme-added group was significantly better than the control group ( Fig.12 ). GME1689_g and GME238_g enzymes were used alone or together in soy sauce fermentation to improve the quality of soy sauce, increase the concentration of amino acids and amino acid nitrogen in soy sauce, and improve the sensory properties of soy sauce.

[0127] In summary, the amino acid sequence provided by the present invention includes an acidic protease, two neutral proteases and an alkaline protease, which have the highest enzyme activity at 45-50°C and have a certain salt tolerance. When applied to soy sauce, the physicochemical indicators, free amino acids, organic acids and flavor substances are better than those of the group without enzyme addition, and have extremely high application prospects in the food field.

[0128] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. Proteases derived from Aspergillus oryzae, respectively named GME1689_g, GME238_g, GME3470_g and GME6033_g, characterized in that: The protease is: (a) a protein consisting of the amino acid sequence shown in SEQ ID No. 1 to SEQ ID No. 4; or, (b) A protein derived from (a) in which the amino acid sequence in (a) is substituted, deleted or added with one or more amino acids and has protease activity.

2. The gene encoding the Aspergillus oryzae-derived protease according to claim 1, characterized in that: A gene sequence consisting of the nucleotides shown in SEQ ID No.5 to SEQ ID No.

8.

3. A recombinant plasmid carrying the gene according to claim 2.

4. The recombinant plasmid according to claim 3, characterized in that The vector of the recombinant plasmid is a pPIC9k vector.

5. A host cell carrying the gene according to claim 2 or the recombinant plasmid according to claim 3 or 4.

6. The host cell according to claim 5, characterized in that The host cell is Pichia pastoris.

7. The method for preparing the protease according to claim 1, characterized in that: The host cell according to claim 5 or 6 is added to the culture medium and cultured until OD 600 =2.0-6.0, methanol is added to the fermentation broth to continue inducing the culture to obtain a fermentation broth containing the protease, and the protease is obtained after separation and purification.

8. Use of the Aspergillus oryzae protease according to claim 1, the gene according to claim 2, the recombinant plasmid according to claim 3 or 4, the host cell according to claim 5 or 6, or the Aspergillus oryzae protease prepared by the preparation method according to claim 7 in bean fermentation products.

9. The use according to claim 8, characterized in that The fermented bean products include but are not limited to fermented black beans, soybean paste, and soy sauce; And / or, the bean paste includes but is not limited to broad bean paste and soybean paste.

10. The use according to claim 9, characterized in that Used for fermentation to prepare soy sauce, Preparation method: When the finished koji is mixed with salt water to start fermentation, the protease derived from Aspergillus oryzae is added in an amount of 100-1000 U / kg, the salinity of the malt is 80-180 g / L, and the fermentation is carried out at 25-40°C.

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