Glucostarch enzyme kega5 and its gene and application

Abstract

The application aims to provide a novel high-yield glucoamylase strain for application in liquor industrial production. Based on the above purpose, the application provides a glucoamylase KeGA5. The amino acid sequence of the glucoamylase KeGA5 is shown as SEQ ID NO. 1. The glucoamylase KeGA5 provided by the application exhibits excellent industrial properties in the exploration of optimal temperature and optimal pH, indicating that the glucoamylase KeGA5 provided by the application can play a role in industrial production such as feed, food, medicine, etc. Specifically, the application uses the recombinant strain transformed with the glucoamylase KeGA5 as a daqu supplement, and the relevant experimental results verify that the recombinant strain transformed with the glucoamylase KeGA5 contributes to the flavor of the liquor body in the brewing of the second batch of liquor, especially in the brewing of the strong-flavor liquor, the recombinant strain transformed with the glucoamylase KeGA5 significantly improves the brewing effect.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of genetic engineering, and particularly relates to a glucose amylase KeGA5 from Daqu, a gene thereof and application. BACKGROUND

[0002] Glucoamylase (EC 3.2.1.3), also known as glucan 1,4-alpha-glucosidase, is an exohydrolase. Glucoamylase has the function of catalyzing the hydrolysis of non-reducing terminal alpha-1,4-glucosidic bonds of soluble starch and related oligosaccharides, and can also catalyze the hydrolysis of alpha-1,6-glucosidic bonds at the branch points of amylopectin, so that free glucose molecules are released at the branch points of amylopectin.

[0003] In the fields of modern biofuel manufacturing, food and beverage processing, cosmetic formulation preparation, feed quality improvement, and harmless treatment of agricultural waste, glucoamylase is widely used as an important enzyme preparation with a huge industrial demand. The catalytic process of glucoamylase is efficient and harmless, and therefore, it has high economic value in the industrial field.

[0004] In the prior art, there are many results of genetic engineering of glucoamylase for saccharifying starch. In traditional liquor production, the solid-state fermentation method mainly uses traditional multi-generation Daqu to complete the liquor-making process by adding the Daqu to starchy raw materials. The preservation and selection of Daqu, which is a major factor affecting the quality of liquor, is the most important step in the liquor-making process. Since the composition of Daqu is complex and the enzyme species is rich, the composition of Daqu that can brew high-quality liquor is also the focus of research by traditional liquor enterprises, and glucoamylase, which is the main force for saccharification of starchy raw materials, has become the focus of research and use in recent years. Gao Yuting et al. excavated and mutagenized high-yield amylase strains in Luzhou-flavor liquor pit mud, and screened an excellent strain TS-5 with high amylase activity. After mutagenesis, a mutant strain TS-5-ARTP with good ability to decompose and utilize cellulose and hemicellulose was obtained.

[0005] Daqu is a traditional comprehensive microbial preparation, which is naturally fermented and plays the functions of saccharifying agent and fermenting agent in Chinese liquor fermentation. The saccharifying power and liquor yield of daqu show a significant positive correlation within a certain range. The fermentation and storage of daqu lasts for 3 to 6 months. According to the top temperature in the fermentation process, daqu can be divided into low-temperature daqu, medium-temperature daqu and high-temperature daqu. Although the three types of daqu all accumulate rich saccharifying enzymes during the fermentation process, the activity of the saccharifying enzymes is highly dependent on the top temperature stage. A higher top temperature will significantly affect the overall saccharifying power of daqu. How to effectively improve the saccharifying power of medium-temperature daqu and high-temperature daqu and reduce the corresponding labor and material costs is a technical problem that the liquor industry focuses on.

[0006] At present, one of the main research focuses in the field of saccharifying enzymes is to modify natural enzymes to obtain enzymes with excellent performance. In the field of traditional liquor, developing enzymes with excellent performance to improve the saccharifying power of daqu is also one of the research focuses in the industry.

[0007] In addition, on the one hand, there are differences in the understanding of those skilled in the art; on the other hand, a large number of literatures and patents have been studied by the applicant when making the present application, but all the details and contents cannot be listed in detail due to the limitation of the space, which does not mean that the present application does not have these characteristics of the prior art. On the contrary, the present application already has all the characteristics of the prior art, and the applicant reserves the right to add relevant prior art in the background art. SUMMARY

[0008] The purpose of the present application is to provide a novel strain of high-yield glucoamylase for use in the production of liquor industry. Based on the above purpose, the present application provides a glucoamylase KeGA5. The amino acid sequence of the glucoamylase KeGA5 is shown in SEQ ID NO. 1 of Table 1. Meanwhile, another purpose of the present application is to provide the application of the glucoamylase KeGA5 in the preparation of daqu for strong-flavor liquor. Preferably, the amino acid sequence of the glucoamylase KeGA5 shown in SEQ ID NO. 1 is added to the preparation of daqu for strong-flavor liquor to improve the flavor of strong-flavor liquor.

[0009] Another purpose of the present application is to provide a glucoamylase gene KeGA5 . The sequence of the glucoamylase gene KeGA5 can encode the glucoamylase KeGA5. Preferably, the nucleotide sequence encoding the glucoamylase KeGA5 is shown in SEQ ID NO. 2 of Table 1. Another purpose of the present application is to provide the application of the glucoamylase gene KeGA5 in the preparation of daqu for strong-flavor liquor. The application of the glucoamylase gene KeGA5 is to add the nucleotide sequence of the glucoamylase KeGA5 shown in SEQ ID NO. 2 or SEQ ID NO. 3 to the preparation of daqu for strong-flavor liquor.

[0010] Another objective of this application is to provide a glucosylamylase gene. KeGA5 The recombinant expression vector. Furthermore, another objective of this application is to include a glucosylamylase gene. KeGA5 The application of recombinant expression vectors in the preparation of strong-aroma baijiu daqu (fermented starter culture). The application of recombinant expression vectors involves adding a gene containing glucosylamylase to the daqu used in the preparation of strong-aroma baijiu daqu. KeGA5 Recombinant expression vectors.

[0011] Another objective of this application is to provide a glucosylamylase gene. KeGA5 The recombinant expression vector pMA5-kega5.

[0012] According to a preferred embodiment, the glucosylamylase gene of this application is... KeGA5 The gene is inserted between suitable restriction enzyme sites on the expression vector, allowing its nucleotide sequence to be operatively linked to the expression regulatory sequence. Preferably, the glucosylase gene is inserted between the NdeI and MluI restriction enzyme sites on plasmid pMA5, placing the nucleotide sequence downstream of and under the regulation of the PHpaⅡ promoter, resulting in the recombinant prokaryotic expression plasmid pMA5-kega5.

[0013] Another objective of this application is to provide a glucosylamylase gene. KeGA5 The recombinant strain. Preferably, the recombinant strain comprises Bacillus subtilis.

[0014] Furthermore, another objective of this application is to include a glucosylamylase gene. KeGA5 The application of recombinant strains in the preparation of strong-aroma baijiu koji (fermented liquor starter). The application of recombinant strains involves adding a gene containing glucosylamylase to the koji used in the preparation of strong-aroma baijiu koji. KeGA5 The recombinant strain comprises Bacillus subtilis and yeast. Particularly preferably, the recombinant strain comprises equal proportions of Bacillus subtilis and yeast.

[0015] Another object of this application is to provide a method for preparing glucosylamylase KeGA5. The method includes the following steps:

[0016] Transform host cells with a recombinant expression vector containing the gene encoding glucoamylase KeGA5;

[0017] Culture host cells to induce the expression of glucoamylase KeGA5;

[0018] The glucosidase KeGA5 was isolated and purified.

[0019] Different from the expression of glucoamylase in the prior art in a eukaryotic expression system (yeast), the present application realizes a new heterologous expression of glucoamylase KeGA5 by using a pMA5 vector and Bacillus subtilis WB600, without adding an inducer in the fermentation process, and the purified glucoamylase can be further obtained by using the method of binding with Ni-NTA gel and gradient elution.

[0020] Unlike eukaryotes, the cell wall and simple structure of prokaryotic cells can withstand drastic temperature changes and survive during heat shock, and the prokaryotic expression system is lower in cost in industrial production. The glucoamylase KeGA5 involved in the present application can be heterologously expressed in a prokaryotic expression system, so that the glucoamylase KeGA5 can be stably produced (transformed starch or expressed) in industrial production, especially in industrial production involving drastic temperature changes.

[0021] According to a preferred embodiment, the host cell can be a host bacterium of a prokaryotic expression system. Preferably, the host bacterium comprises Escherichia coli, Bacillus, Streptomyces or cyanobacteria. Particularly preferably, the host bacterium comprises Bacillus subtilis (B. subtilis) (B. subtilis). Bacillus subtilis )。

[0022] According to a preferred embodiment, the host cell can be a host bacterium of a eukaryotic expression system. Preferably, the host bacterium comprises yeast. Particularly preferably, the host bacterium comprises Pichia pastoris (P. pastoris), Saccharomyces cerevisiae (S. cerevisiae) or Hansenula polymorpha (H. polymorpha). Pichia pastoris )。 Saccharomyces cerevisiae )。 Hansenula polymorpha )。

[0023] Another purpose of the present application is to provide a Daqu. Preferably, the provided Daqu is used for the brewing of Luzhou-flavor Chinese liquor. Preferably, the Daqu comprises a recombinant strain of transformed glucoamylase KeGA5.

[0024] Another purpose of the present application is to provide the application of the glucoamylase KeGA5 or the above-mentioned Daqu containing the glucoamylase KeGA5 in the field of feed, food or medicine for the purpose of diagnosis and treatment of non-disease. For example, the preparation of glucose for glucose injection.

[0025] Another purpose of the present application is to provide a method for preparing Luzhou-flavor Chinese liquor. The method comprises the following steps: using the above-mentioned Daqu to prepare Luzhou-flavor Chinese liquor.

[0026] According to a preferred embodiment, the method comprises the following steps: the recombinant strain added to the Daqu used for preparing Luzhou-flavor Chinese liquor comprises an equal proportion of Bacillus subtilis and yeast. Both the Bacillus subtilis and the yeast are Bacillus subtilis and yeast transformed with glucoamylase KeGA5.

[0027] According to a preferred embodiment, the method comprises the following step: during the preparation fermentation, 2 mL of the recombinant strain bacterial solution with a viable bacterial count of more than 120 per milliliter is mixed with each kilogram of the preparation.

[0028] Another object of the present application is to provide a glucoamylase KeGA5 derived from Clostridium phytofermentans. Kroppenstedtia

[0029] Another object of the present application is to provide a glucoamylase KeGA5 capable of improving enzyme activity under the treatment of low concentration (1 mM) of Co 2+ , high concentration (10 mM) of Co 2+ , high concentration (10 mM) of Mg 2+ . In particular, the glucoamylase KeGA5 involved in the present application is suitable for decomposing starch wastewater containing a large amount of metal ions (e.g., cobalt, magnesium, calcium). Meanwhile, another object of the present application is to provide a metal ion Co 2+ specifically catalyzing the glucoamylase KeGA5.

[0030] The optimal pH of the glucoamylase KeGA5 of the present application is 7.0-8.0. The glucoamylase KeGA5 of the present application can maintain an enzyme activity level of more than 78% after being treated in an environment with a pH of 4.0-9.0 for 30 min.

[0031] The optimal temperature of the glucoamylase KeGA5 of the present application is 55°C. The enzyme activity of the glucoamylase KeGA5 of the present application is almost not lost (98.27%-100%) after being treated in an environment with a temperature of 45°C or below for 30 min, and it can maintain an enzyme activity level of more than 81% after being treated at 50°C for 4 h. The above experimental results show the excellent temperature stability of the glucoamylase KeGA5 of the present application.

[0032] The glucoamylase KeGA5 provided by the present application exhibits excellent industrial properties in the exploration of optimal temperature and optimal pH, indicating that the glucoamylase KeGA5 provided by the present application can play a role in the industrialized production of feed, food, medicine, etc. Specifically, the purpose of producing a glucoamylase with excellent properties and suitable for industrial application by using genetic engineering means can be achieved according to the technical solution of the present application, and the glucoamylase is produced by using genetic engineering means for industrialization, so that the engineered glucoamylase KeGA5 plays a role in the brewing industry, especially in the brewing of strong-flavor liquor, to improve the brewing effect.

[0033] ​Further, the application further proves the components in high-quality Daqu, which has an important role for preparing Daqu used for brewing Luzhou-flavor liquor. The glucoamylase KeGA5 of the application can be produced by genetic engineering, and when it is used in the saccharification process in the liquor production scene, on the one hand, it can maintain the homogeneity of Daqu as a traditional fermentation agent, and on the other hand, it avoids the corresponding enzyme activity loss because it does not actually experience the top temperature stage in the Daqu fermentation process, and is more suitable for playing a high-efficiency saccharification performance.

[0034] A further purpose of the application is to provide a preparation method of Luzhou-flavor liquor:

[0035] The mixture is fermented. Preferably, the Bacillus subtilis bacterial liquid with the viable count of more than 120 per milliliter of the glucoamylase KeGA5 is added in an amount of 5-10 mL. Preferably, the Bacillus subtilis bacterial liquid with the viable count of more than 120 per milliliter of the glucoamylase KeGA5 is added in an amount of 10-20 mL. Preferably, the Bacillus subtilis bacterial liquid with the viable count of more than 120 per milliliter of the glucoamylase KeGA5 is added in an amount of 20-30 mL. Preferably, the Bacillus subtilis bacterial liquid with the viable count of more than 120 per milliliter of the glucoamylase KeGA5 is added in an amount of 30-50 mL. More preferably, the Bacillus subtilis bacterial liquid with the viable count of more than 120 per milliliter of the glucoamylase KeGA5 is added in an amount of 5 mL. The Bacillus subtilis bacterial liquid with the viable count of more than 120 per milliliter of the glucoamylase KeGA5 is added in an amount of 10 mL. The Bacillus subtilis bacterial liquid with the viable count of more than 120 per milliliter of the glucoamylase KeGA5 is added in an amount of 15 mL. The Bacillus subtilis bacterial liquid with the viable count of more than 120 per milliliter of the glucoamylase KeGA5 is added in an amount of 20 mL.

[0036] According to a preferred embodiment, the fermentation time can be 10-30 days. Preferably, the fermentation time can be 20-30 days. Preferably, the fermentation time can be 25-30 days. More preferably, the fermentation time can be 27 days. More preferably, the fermentation time can be 25 days. More preferably, the fermentation time can be 26 days.

[0037] To verify the impact of the glucosylase KeGA5, screened from high-quality Daqu (a type of starter culture), on the quality of Baijiu (Chinese liquor), the applicant designed a control experiment (including four schemes: high-temperature Daqu, high-temperature Daqu + yeast culture with converted glucosylase KeGA5, high-temperature Daqu + Bacillus subtilis culture with converted glucosylase KeGA5, and high-temperature Daqu + yeast culture with converted glucosylase KeGA5 + Bacillus subtilis culture with converted glucosylase KeGA5) to verify that Baijiu brewed using high-temperature Daqu + yeast culture with converted glucosylase KeGA5 + Bacillus subtilis culture with converted glucosylase KeGA5 as inoculum for the second batch of mash is of excellent quality, with a mellow, sweet, and full-bodied flavor and a long finish. This application obtained a prokaryotic fermentation system based on the screening of strains that convert glucosylase KeGA5. Using this fermentation system with high-temperature Daqu to prepare strong-aroma Baijiu can reduce fermentation time, increase fermentation efficiency, and significantly improve the flavor of strong-aroma Baijiu. Attached Figure Description

[0038] Figure 1 The recombinant expression vector pMA5-kega5 provided by this invention;

[0039] Figure 2 The SDS-PAGE protein electrophoresis pattern of glucoamylase KeGA5 provided by this invention includes: a molecular weight marker (pre-stained protein marker); lane 1 is the supernatant of Bacillus subtilis WB600 lysate containing empty vector plasmid pMA5; lane 2 is the supernatant of Bacillus subtilis WB600 lysate containing recombinant plasmid pMA5-kega5; lane 3 is nickel column flow-through buffer; lane 4 is 10 mM imidazole elution buffer; lane 5 is 20 mM imidazole elution buffer; lane 6 is 50 mM imidazole elution buffer; lane 7 is 80 mM imidazole elution buffer; lane 8 is 100 mM imidazole elution buffer; lane 9 is 200 mM imidazole elution buffer; and lane 10 is 500 mM imidazole elution buffer.

[0040] Figure 3 A statistical chart showing the optimal pH of the glucoamylase KeGA5 provided by this invention;

[0041] Figure 4 A statistical chart showing the optimal temperature for the glucoamylase KeGA5 provided by this invention;

[0042] Figure 5 The activity statistics of glucoamylase KeGA5 provided by the present invention after incubation at different pH values ​​for 30 min;

[0043] Figure 6 A statistical graph showing the activity of the glucoamylase KeGA5 provided by this invention after incubation at different temperatures for 30 min.

[0044] Figure 7 The statistical chart of the activity dynamic change of the glucose amylase KeGA5 provided by the present application at different time points of the enzyme incubated at different temperatures for 0-6 h;

[0045] Figure 8 The statistical chart of the influence of different metal ions and chemical reagents on the activity of the glucose amylase KeGA5 provided by the present application. DETAILED DESCRIPTION

[0046] The present application will be described in detail below with reference to the accompanying drawings.

[0047] Unless otherwise defined, all scientific and technical terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description of the application herein is for describing specific embodiments only and is not intended to be limiting of the application. The use of the terms "and / or" includes a combination of one or more of the associated listed items. The above described terms are understood by those of ordinary skill in the art according to the specific context in which they are used.

[0048] The experimental methods in the following examples are all conventional methods, and are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions, unless otherwise specified. If not specifically indicated, the examples are carried out according to the conventional experimental conditions or the conditions suggested by the manufacturer's instructions. The raw materials and reagents used in the present application are commercially available, and any biological germplasm material can be provided to the public as scientific research.

[0049] Compared with the conventional use of exogenous saccharifying enzymes, in-depth analysis of the saccharifying enzyme spectrum of Daqu and targeted selection of saccharifying enzymes from the saccharifying enzyme library of Daqu itself provide a more optimal customized selection strategy for liquor production. Although traditional microbial culture methods can screen a certain amount of enzyme-producing strains, most microorganisms in Daqu are difficult to culture under laboratory conditions, and metagenomics and heterologous expression technology breaks through such operational barriers, and is expected to maximize the excavation of active enzyme genes in nature and obtain potential and novel glucose amylases.

[0050] Based on the above purposes, on the basis of the functional annotation results of multi-omics data, a new glucose amylase coding gene fragment is obtained by primer design and target fragment amplification means using Daqu metagenomic DNA as a template, and is named KeGA5 Homology comparison finds that it only has a match with the whole genome sequence (3564999 bp in length) of Kroppenstedtia Ivorensis Kroppenstedtia eburnea ​

[0051] C. propinquus Kroppenstedtia ) as a class of heat-resistant, aerobic, spore-forming gram-positive bacteria, are distributed in various Daqu (for example: Xiangangxing et al. compared and analyzed the microbial community structure and physicochemical properties of different grades of Luzhou-flavor Daqu in Food Science, and found that the dominant bacterial genera in the two groups of Daqu samples, superior Daqu and first-grade Daqu, contained C. propinquus). Previous studies have found that glucose amylase produced by this type of microorganism can continue to the early fermentation stage (0 to 12 days) of fermented grains, and has strong application and conversion potential in the saccharification process of liquor production. However, so far, this type of microorganism has rarely been directly isolated from Daqu systems.

[0052] The glucose amylase KeGA5 of the present application belongs to the glycoside hydrolase GH15 family and is derived from Daqu. The glucose amylase KeGA5 of the present application exhibits an enzyme activity level of more than 93% in the range of 50°C to 55°C, which is also the commonly used temperature range for liquor fermentation. In the pH range of 6.0 to 8.0, the glucose amylase KeGA5 of the present application can exhibit an enzyme activity level of more than 82%. Through relevant property experiments, it can be known that the glucose amylase KeGA5 of the present application is suitable for the methods of solid-state fermentation, liquid-state fermentation and solid-liquid fermentation of liquor.

[0053] The present application provides a novel glucose amylase KeGA5. Since the gene fragment expressing the glucose amylase KeGA5 is directly amplified from the metagenomic DNA of Daqu, based on the genetic KeGA5 The glucose amylase expressed as a component for promoting starch decomposition can maintain the homogeneity of Daqu as a traditional fermentation agent when directly used as a Daqu component.

[0054] The results of homology alignment show that the glucose amylase KeGA5 is produced by C. propinquus, and there is currently no related report on the development of enzyme proteins of this type of bacteria.

[0055] C. propinquus belongs to the order of Bacillales, and therefore the gene sequence is derived from bacteria of the order of Bacillales, which also indicates that the glucose amylase expressed by the gene KeGA5 The glucose amylase expressed is easy to realize heterologous and soluble high expression in hosts such as B. subtilis, and the expression process is simple to operate without the need for additional induction.

[0056] In the study of the properties of the glucose amylase KeGA5, it was found that the glucose amylase KeGA5 has high temperature stability and has application and development potential in the fields of food and fermentation. Example 1

[0057] This example relates to the cloning of the glucose amylase KeGA5 coding gene.

[0058] According to the results of the metagenomic and metaproteomic sequencing of the high-temperature Daqu (fermentation top temperature 65℃) collected in the laboratory, a bacterial-derived glucoamylase was screened, and the coding gene thereof was 1974 bp in length. The primers were designed using the gene, and the primer sequences are SEQ ID NO. 3 and SEQ ID NO. 4 shown in Table 1.

[0059] The total DNA extracted from the high-temperature Daqu was used as a template to amplify the target fragment using the above primers. The PCR reaction parameters were as follows: 95℃ for 5 min; 95℃ for 30 s, 56℃ for 45 s, 72℃ for 100 s, 34 cycles; 72℃ for 10 min. After electrophoresis detection, the PCR amplification product band was bright and single, and the PCR product purification kit was used for purification, and then sequencing was performed, and the target gene was obtained, and the sequence is SEQ ID NO. 2 shown in Table 1.

[0060] The above gene encodes a protein of 658 amino acids, and the theoretical molecular weight is 74.04 kDa, and the amino acid sequence is SEQ ID NO. 1 shown in Table 1. Example 2

[0061] This example relates to the expression of the glucoamylase KeGA5 coding gene in Bacillus subtilis.

[0062] The purified product was used as a new PCR amplification template, and the primer pair was used for secondary amplification, and the primer sequences are SEQ ID NO. 5 and SEQ ID NO. 6 shown in Table 1.

[0063] SEQ ID NO. 6 contains a 6×His tag, and the sequence of the 6×His tag is SEQ ID NO. 7 shown in Table 1.

[0064] After the new PCR product was purified by gel recovery, it was connected with the linearized pMA5 vector digested by NdeI and MluI according to the operation steps of the homologous recombination kit (ClonExpress II OneStep Cloning Kit, Novagen), and the connection product was transformed into E. coli DH5α competent cells, and the recombinant bacteria were screened on LB plates containing ampicillin. The plasmid DNA extraction kit (FastPure Plasmid Mini Kit, Novagen) was used to extract the plasmid, and the vector construction was verified by sequencing to be correct, and the constructed recombinant expression vector pMA5-kega5 is shown in Table 1. The recombinant plasmid was transformed into Bacillus subtilis WB600 competent cells by chemical transformation method, and the recombinant bacteria were screened on LB plates containing kanamycin. After 37℃ culture overnight, the positive clones were identified by colony PCR, and the successful engineering bacteria were obtained by sequencing. Figure 1 ​B.subtilis WB600-pMA5-kega5.

[0065] Picking B. subtilis WB600-pMA5-kega5 monoclonal to LB medium (containing 50 μg / mL kanamycin), 37°C 220 rpm shaker culture for 8~12 h, then the seed liquid is inoculated into TB medium (containing 50 μg / mL kanamycin) at 2% inoculation amount, 37°C 220 rpm shaker culture for 24 h. The seed liquid contains the above culture solution containing a large number of live bacteria.

[0066] Centrifuge the bacterial solution at 8000 rpm for 10 min, and take the precipitate. The precipitate is washed twice with sterile PBS solution, and then 100 μL of 20 mg / mL lysozyme is added and incubated at 37°C 220 rpm shaker for 30 min.

[0067] Ultrasonic crushing is carried out on the bacterial solution in an ice bath, and the ultrasonic program is: 350 W, working for 4 s~stopping for 6 s cycle, total time 45 min.

[0068] Centrifuge at 8000 rpm for 20 min at 4°C, and take the supernatant through a 0.22 μm PES filter membrane to obtain the crude enzyme solution of glucose amylase KeGA5. Example 3

[0069] This example relates to the purification of glucose amylase KeGA5.

[0070] The crude enzyme solution is subjected to affinity chromatography by Ni-NTA gravity column (Shanghai Sangon), and the target protein is separated and purified by using buffer with different imidazole gradients (50 mM NaH2PO4, 300 mM NaCl, 10~500 mM imidazole, pH 8.0). The buffer with different imidazole gradients refers to the buffer with only the concentration of imidazole changed as Figure 2 The concentration gradient shown indicates that the other components and their concentrations remain unchanged.

[0071] SDS-PAGE protein electrophoresis is used to detect the purification effect of the enzyme, and the results are shown in Figure 2 The target protein band is single, and the size is consistent with the theory, proving that pure enzyme protein is obtained.

[0072] All eluate containing the target enzyme protein is combined by using an ultrafiltration tube with a molecular weight cutoff of 30 kDa (Amicon Ultra-15, Millipore), concentrated at 4°C 4000 rpm and imidazole and impurities are removed, and the removal effect of impurities is detected by Bradford solution. The protein in the pure enzyme solution is quantified by BCA method at 562 nm wavelength. Example 4

[0073] This example relates to the property analysis of glucose amylase KeGA5.

[0074] DNS method was used to analyze the activity of glucose amylase KeGA5. 100 μL of 50 μg / mL enzyme solution was added to 900 μL of 1% soluble starch solution preheated to 55°C, and reacted at 55°C for 20 min. The reaction was terminated by adding 1.5 mL of DNS solution, boiled in a water bath for 6 min, cooled in an ice bath to room temperature, and the absorbance was measured at 540 nm.

[0075] The definition of glucose amylase activity unit: the amount of enzyme required to catalyze the hydrolysis of substrate to release 1 μmol of reducing sugar per minute is one enzyme activity unit (U).

[0076] (1) Optimum pH detection

[0077] The enzyme activity of glucose amylase KeGA5 was measured under different pH (2.0-11.0) conditions to confirm its optimum reaction pH, and the specific activity of the enzyme at the optimum pH was defined as 100%. As shown in Figure 3 , the enzyme activity of glucose amylase KeGA5 reached the highest level at pH 7.0-8.0, indicating that the optimum reaction pH of glucose amylase KeGA5 was 7.0-8.0. Glucose amylase KeGA5 could maintain more than 82% of the enzyme activity level at pH 6.0. The relative enzyme activity measurement method was used in this application.

[0078] (2) Optimum temperature detection

[0079] The enzyme activity of glucose amylase KeGA5 was measured under different temperatures (10-80°C) to confirm its optimum reaction temperature, and the specific activity of the enzyme at the optimum temperature was defined as 100%. As shown in Figure 4 , the enzyme activity of glucose amylase KeGA5 reached the highest level at 55°C, indicating that the optimum reaction temperature of glucose amylase KeGA5 was 55°C.

[0080] (3) pH stability detection

[0081] Glucose amylase KeGA5 was incubated at different pH (2.0-11.0) conditions for 30 min, and then its enzyme activity was detected at the optimum pH and optimum temperature conditions, and the highest specific activity after incubation was defined as 100%. As shown in Figure 5 , glucose amylase KeGA5 could maintain more than 78% of the enzyme activity level when treated at pH 4.0-9.0 for 30 min.

[0082] (4) Thermal stability detection

[0083] glucoamylase KeGA5 was incubated at different temperatures (20-90°C) for 30 min, and then its enzyme activity was detected under the optimal pH and temperature conditions. The highest enzyme specific activity after incubation was defined as 100%. As shown in Table 1, the enzyme activity of glucoamylase KeGA5 after incubation at 20°C was 99.07%, the enzyme activity after incubation at 30°C was 98.27%, the enzyme activity after incubation at 40°C was 100%, the enzyme activity after incubation at 45°C was 99.87%, and the enzyme activity after incubation at 50°C was 93.61%. The results showed that the enzyme activity of glucoamylase KeGA5 was almost not lost (98.27%-100%) when it was treated at 45°C or below for 30 min. Figure 6

[0084] glucoamylase KeGA5 was incubated at different temperatures (50°C, 55°C, and 60°C) for 0 min, 2 min, 5 min, 30 min, 60 min, 120 min, 180 min, 240 min, 300 min, and 360 min, and then its enzyme activity was detected under the optimal pH and temperature conditions. The results are shown in Table 2. The glucoamylase KeGA5 maintained more than 81% of the enzyme activity level after being treated at 50°C for 4 h, showing excellent temperature stability. Figure 7

[0085] (5) Effects of metal ions and chemical reagents

[0086] 200 μL of 50 μg / mL glucoamylase KeGA5 sample was taken, and 10 mM and 1 mM of metal ions or chemical reagents (NaCl, NH4Cl, NiCl2, KCl, CaCl2, CuSO4, CoCl2, FeCl2, MnCl2, BaCl2, ZnSO4, MgCl2, FeCl3, AlCl3, SDS, and EDTA, a total of 16 kinds) were added, respectively, and then incubated at 37°C for 30 min. The enzyme activity was detected under the optimal pH and temperature conditions. The control sample was subjected to the same incubation and dilution, but no metal ions and chemical reagents were added, and the enzyme activity was set to 100%. As shown in Table 3, the enzyme activity of the glucoamylase KeGA5 treated with 1 mM CoCl2was 164%, which was significantly higher than that of the control group. Figure 8 2+ The results showed that low-concentration (1 mM) CoCl2could significantly improve the enzyme activity of the glucoamylase KeGA5, and the relative enzyme activity level was as high as 164%. 2+

[0087] As shown in Table 4, the enzyme activity of the glucoamylase KeGA5 treated with 1 mM CoCl2was 164%, which was significantly higher than that of the control group. Figure 8 ​​​​As shown, at high concentrations (10 mM) of Co 2+ Under treatment, the activity level of glucoamylase KeGA5 was 100.70%. This result indicates that high concentrations (10 mM) of Co... 2+ This promoting effect was not observed.

[0088] like Figure 8 As shown, at high concentrations (10 mM) of Mg 2+ Under treatment, the activity level of glucoamylase KeGA5 was 105.16%. This result indicates that high concentrations (10 mM) of Mg... 2+ It also has a certain promoting effect on enzyme activity, but the effect is weak.

[0089] like Figure 8 As shown, in 1 mM and 10 mM Ni + Under the treatment, the activity levels of glucoamylase KeGA5 were 100.42% and 47.84%, respectively; in 1 mM and 10 mM Cu... 2+ Under the treatment, the activity levels of glucoamylase KeGA5 were 22.73% and 0%, respectively; at 1 mM and 10 mM Fe... 2+ Under the treatment, the activity levels of glucoamylase KeGA5 were 82.29% and 24.69%, respectively; at 1 mM and 10 mM Mn 2+ Under the treatment, the activity levels of glucoamylase KeGA5 were 78.24% and 0%, respectively; in 1 mM and 10 mM Zn 2+ Under the treatment, the activity levels of glucoamylase KeGA5 were 60.25% and 21.48%, respectively; in 1 mM and 10 mM Fe... 3+ Under the treatment, the activity levels of glucoamylase KeGA5 were 79.64% and 0%, respectively; at 1 mM and 10 mM Al 3+ Under the first treatment, the activity levels of glucoamylase KeGA5 were 84.80% and 1.53%, respectively; under 1 mM and 10 mM SDS treatment, the activity levels of glucoamylase KeGA5 were 82.57% and 49.93%, respectively; and under 1 mM and 10 mM EDTA treatment, the activity levels of glucoamylase KeGA5 were 94.98% and 67.22%, respectively.

[0090] The results show that Ni + (1 mM), Cu 2+ (1 mM, 10 mM), Fe 2+ (1 mM, 10 mM), Mn 2+ (1 mM, 10 mM), Zn 2+ (1 mM, 10 mM), Fe 3+(1 mM, 10 mM), Al 3+ (1 mM, 10 mM), SDS (1 mM, 10 mM), EDTA (1 mM, 10 mM) showed certain inhibition on the KeGA5 enzyme activity, wherein high concentration (10 mM) of Cu 2+ , Mn 2+ , Fe 3+ , Al 3+ almost completely inhibited the KeGA5 enzyme activity. Example 5

[0091] This example relates to experiments and effect evaluation for improving liquor quality by using yeasts and Bacillus subtilis containing glucose amylase KeGA5.

[0092] After obtaining glucose amylase KeGA5 capable of being expressed in a prokaryotic system from Daqu, the applicant inoculated prokaryotic microorganisms containing glucose amylase KeGA5 and / or eukaryotic microorganisms containing glucose amylase KeGA5 as a supplement into second-crop fermented grains, and explored the effects of prokaryotic microorganisms containing glucose amylase KeGA5 and / or eukaryotic microorganisms on the production of Luzhou-flavor liquor.

[0093] The recombinant plasmid shown in Example 2 was used to transform Bacillus subtilis WB600 competent cells by a chemical transformation method, and the recombinant bacteria were screened on LB plates containing kanamycin. The recombinant plasmid of pYES2 / NT and KeGA5 was constructed by homologous recombination, and the constructed plasmid was transformed into Saccharomyces cerevisiae NMY51 competent cells by a chemical transformation method, and the recombinant bacteria were screened on SD medium.

[0094] YPD liquid medium and LB liquid medium were used for cultivation, and the bacteria were cultured at 37°C and obtained a viable count of more than 120 per milliliter of bacteria.

[0095] The bacteria liquid obtained by the above scale-up cultivation was inoculated into 50 kg of second-crop fermented grains according to the following scheme, and the fermented grains were fermented for 27 days, and then the base liquor was obtained and subjected to taste evaluation.

[0096] The inoculation scheme included: A scheme of 10 kg high-temperature Daqu; B scheme of 10 kg high-temperature Daqu + 10 mL of glucose amylase KeGA5 yeast liquid; C scheme of 10 kg high-temperature Daqu + 10 mL of glucose amylase KeGA5 Bacillus subtilis liquid; D scheme of 10 kg high-temperature Daqu + 5 mL of glucose amylase KeGA5 Bacillus subtilis liquid + 5 mL of glucose amylase KeGA5 yeast liquid.

[0097] The high-temperature Daqu is a kind of amylase fermentation agent prepared under a high-temperature condition of 50℃ or above for fermenting the second fermentation mash in the present embodiment.

[0098] Inoculation method: 10 mL of the bacterial solution capable of converting the glucose amylase KeGA5 was evenly divided into four portions, and was poured into the second fermentation mash in batches on the first day, the third day, the fifth day and the tenth day of fermentation.

[0099] The experimental results are shown in Table 2. According to the relevant results, when the glucose amylase KeGA5 converted by the prokaryotic system and the glucose amylase KeGA5 converted by the eukaryotic system are used as the supplements and are used together with the high-temperature Daqu to act on the second fermentation mash, the taste of the produced liquor is sweet and full at the entrance, and the aftertaste is long, and the quality is higher. Therefore, the raw materials containing the second fermentation mash can improve the brewing efficiency after being inoculated with the Daqu containing the glucose amylase KeGA5 converted by the prokaryotic system and the glucose amylase KeGA5 converted by the eukaryotic system. Here, the raw materials refer to the grains used for brewing liquor.

[0100] Table 1

[0101]

[0102]

[0103]

[0104] Table 2

[0105]

[0106] It should be noted that the above specific embodiments are exemplary, and those skilled in the art can think of various solutions under the inspiration of the disclosure of the present application, and these solutions also belong to the disclosed range of the present application and fall within the protection scope of the present application. Those skilled in the art should understand that the specification and drawings of the present application are illustrative and not constitute a limitation on the claims. The protection scope of the present application is defined by the claims and their equivalents. The specification of the present application contains multiple inventive concepts, such as “preferably”, “according to a preferred embodiment” or “optionally”, which all indicate that the corresponding paragraph discloses an independent concept, and the applicant reserves the right to file a divisional application according to each inventive concept. Throughout the text, the features introduced by “preferably” are only optional ways, and should not be understood as necessarily set, therefore the applicant reserves the right to abandon or delete the relevant preferred features at any time.

Claims

1. A type of starter culture for preparing strong-aroma baijiu, characterized in that, The Daqu contains a recombinant strain of glucoamylase KeGA5 with the amino acid sequence shown in SEQ ID NO.

1.

2. The Daqu (a type of Chinese liquor) according to claim 1, characterized in that, The recombinant strain added to the daqu (fermentation starter) used to prepare strong-aroma baijiu also includes Bacillus subtilis and yeast.

3. The Daqu (a type of Chinese liquor) according to claim 2, characterized in that, The recombinant strain added to the daqu (fermentation starter) used to prepare strong-aroma baijiu contains equal proportions of Bacillus subtilis and yeast.

4. The application of glucoamylase KeGA5 in the preparation of strong-aroma baijiu daqu (fermentation starter), characterized in that... The amino acid sequence of the glucoamylase KeGA5 added to the preparation of strong-aroma baijiu daqu is shown in SEQ ID NO. 1, or its nucleotide sequence is shown in SEQ ID NO.

2.

5. A method for preparing strong-aroma baijiu, characterized in that, The method includes the following steps: Using a gene containing inverted glucosylamylase KeGA5 The recombinant strain of Daqu was used to prepare strong-aroma Baijiu. The amino acid sequence of the glucoamylase KeGA5 is shown in SEQ ID NO.1 or its nucleotide sequence is shown in SEQ ID NO.

2.

6. The method for preparing strong-aroma baijiu according to claim 5, characterized in that, The recombinant strain added to the daqu (fermentation starter) used to prepare strong-aroma baijiu contains Bacillus subtilis and / or yeast.

7. The method for preparing strong-aroma baijiu according to claim 6, characterized in that, The recombinant strain added to the daqu (fermentation starter) used to prepare strong-aroma baijiu contains equal proportions of Bacillus subtilis and yeast.

8. The method for preparing strong-aroma baijiu according to claim 7, characterized in that, During the preparation and fermentation process, each kilogram of prepared material is mixed with 2 mL of recombinant bacterial solution containing more than 120 live bacteria per milliliter.

9. The use of the Daqu (a type of starter culture) as described in claim 1 in the preparation of feed, food, or medicine for non-disease diagnostic and therapeutic purposes.

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