CN111088199B

CN111088199B - Streptomyces fuselaphus and application thereof in production of proteolytic enzyme

Info

Publication number: CN111088199B
Application number: CN202010095218.5A
Authority: CN
Inventors: 吴敬; 颜正飞; 秦琴
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2020-02-14
Filing date: 2020-02-14
Publication date: 2021-10-19
Anticipated expiration: 2040-02-14
Also published as: CN111088199A

Abstract

The invention discloses streptomyces glaucescens and application thereof in production of proteolytic enzyme, and belongs to the technical field of microorganisms and fermentation. The invention screens out a streptomyces darkling T20, the streptomyces darkling T20 can efficiently degrade protein, the streptomyces darkling T20 is coated on a solid culture medium containing 1g/L rice protein to be cultured for 48h, and the diameter of a transparent ring of the solid culture medium can reach 25 mm. The invention screens out a streptomyces darkling T20, the streptomyces darkling T20 can produce a proteolytic enzyme with a high-yield amino acid sequence shown as SEQ ID NO.2, and the streptomyces darkling T20 is inoculated into a fermentation culture medium for fermentation for 48 hours, so that the enzyme activity of the proteolytic enzyme in a fermentation supernatant can reach 1500U/mL.

Description

Streptomyces fuselaphus and application thereof in production of proteolytic enzyme

Technical Field

The invention relates to streptomyces cinerea and application thereof in production of proteolytic enzyme, and belongs to the technical field of microorganisms and fermentation.

Background

Compared with proteins such as soybean protein, whey protein and the like, the rice protein is recognized as high-quality protein, has low content of nutrition inhibiting factors, has no anaphylactic reaction, contains amino acids required by a human body, has reasonable amino acid proportion, has far-reaching biological value, is comparable with the biological value of shrimps, beef and the like, and accords with an ideal mode recommended by the World Health Organization (WHO). After the rice protein is hydrolyzed into short peptide, the short peptide nutrient solution with higher nutrient value can be prepared, and the short peptide nutrient solution can be used as a high-value additive in the industries of health care products, beverages, cosmetics and the like.

Among rice peptides, small-molecule peptides having a molecular weight of less than 1000Da, which are collectively called "small-molecule peptides", can be rapidly absorbed by The small intestine of a human body, not only do not cause side effects such as overnutrition, but also some specific small-molecule peptides have unique physiological functions such as antioxidant ability (specifically, see references: Yan et al, Food Chemistry,2015,179: 290) improving piglet intestinal flora (specifically, see references: Yanya Entensing, Nanchang university [ M ],2018), Angiotensin Converting Enzyme (ACE) inhibitory activity (specifically, see references: Wang et al, LWT-Food Sci Technology,2017,75:93-99), and anticancer activity (specifically, see references: Kannan et al, The Open biological sources Journal,2009,2:17-20), etc., and are key indicators of The quality of rice peptides, the market competitiveness of the rice peptide product can be greatly improved by increasing the content of the small molecular peptide.

At present, the preparation method of the small molecular peptide mainly comprises an enzymolysis method, a microbial fermentation method, an acid-base hydrolysis method, a chemical synthesis method and the like, wherein the enzymolysis method is an industrially feasible method with high safety and environmental protection. However, the conventional enzymatic hydrolysis method still has many defects, for example, the preparation method described in the patent publication No. CN1102229643A can make the content of small molecule peptides in the obtained rice peptide product reach about 50%, but it needs to use multiple enzyme preparations for synergistic enzymatic hydrolysis in the preparation process, and it needs to use a hollow fiber membrane, which greatly prolongs the production period of the rice peptide product and increases the industrial production cost of the rice peptide product; patent No. CN103440949A firstly adopts the steps of crushing, size mixing, washing, size mixing, reacting, concentrating, dehydrating and the like to pretreat the rice dregs, and then adopts a plurality of proteolytic enzymes to cooperatively treat the pretreated rice dregs, however, the method mainly adopts the pretreatment mode to improve the content of the small molecular peptides in the rice peptide products, the content of the small molecular peptides in the rice peptide products does not change obviously before and after the enzymolysis process, and the method has the disadvantages of complicated preparation steps, large energy consumption, large enzyme adding amount and is not beneficial to the industrialization process.

Therefore, there is an urgent need to find a method for preparing rice peptide with simple preparation process, short preparation period, low cost and high content of small molecule peptide in the prepared rice peptide to overcome the above-mentioned disadvantages.

Disclosure of Invention

[ problem ] to

The technical problem to be solved by the invention is to provide the method for preparing the rice peptide, which has the advantages of simple preparation process, short preparation period, low cost and high content of the small molecular peptide in the prepared rice peptide.

[ solution ]

In order to solve the problems, the invention provides Streptomyces glaucous (Streptomyces canus) T20, wherein the Streptomyces glaucous (Streptomyces canus) T20 is preserved in China center for type culture collection in 11 months and 15 days in 2019, the preservation number is CCTCC NO: M2019936, and the preservation address is Wuhan, Wuhan university in China.

The Streptomyces griseus (Streptomyces canus) T20 is derived from soil samples in a flour mill plant area of a lake and coast area of Wuxi city, Jiangsu province, the strain is analyzed by sequencing, the 16SrDNA sequence of the strain is shown as SEQ ID NO.1, the sequence obtained by sequencing is compared with the nucleic acid sequence in Genbank, the similarity of the 16SrDNA sequence and the 16S rDNA sequence of the Streptomyces griseus (Streptomyces canus) DSM 40017 is up to 98.3 percent, the strain with high similarity is constructed into a phylogenetic tree (particularly shown in figure 1), and the result shows that the strain and the Streptomyces griseus (Streptomyces canus) cfcc 3169 belong to the same branch and are named as Streptomyces griseus (Streptomyces canus) T20.

The colony of the Streptomyces cinerea (Streptomyces canus) T20 on the LB solid culture medium is small, white, regular in edge, circular and 2-4 mm in diameter.

The growth temperature of the Streptomyces glaucescens (Streptomyces canus) T20 is 20-45 ℃, and the optimal growth temperature is 30 ℃.

The growth pH of the Streptomyces glaucescens (Streptomyces canus) T20 is 6.0-8.0, and the optimal growth pH is 7.0.

The Streptomyces glaucescens (Streptomyces canus) T20 can grow in a culture medium containing 5g/LNCl and has certain salt tolerance.

The invention also provides application of the Streptomyces nigrescens (Streptomyces canus) T20 in producing proteolytic enzyme.

The invention also provides a method for producing the proteolytic enzyme, which comprises the steps of inoculating the Streptomyces nigrolae (Streptomyces canus) T20 into a fermentation medium for fermentation to obtain fermentation liquor containing the proteolytic enzyme, and then extracting the fermentation liquor to obtain the proteolytic enzyme.

In one embodiment of the invention, the amino acid sequence of the proteolytic enzyme is shown in SEQ ID No. 2.

In one embodiment of the invention, the nucleotide sequence of the gene encoding the proteolytic enzyme is shown in SEQ ID No. 3.

In one embodiment of the invention, the components of the fermentation medium comprise rice protein powder, sodium dihydrogen phosphate, disodium hydrogen phosphate, magnesium sulfate and ferrous sulfate.

In one embodiment of the invention, the components of the fermentation medium comprise 2.5g/L rice protein powder, 0.1g/L sodium dihydrogen phosphate, 0.1g/L disodium hydrogen phosphate, 0.05g/L magnesium sulfate and 0.001g/L ferrous sulfate.

In one embodiment of the present invention, the fermentation temperature is 20 to 45 ℃, the rotation speed is 100 to 200rpm, and the pH is 6.0 to 8.0.

The invention also provides application of the Streptomyces nigrescens (Streptomyces canus) T20 in producing protein peptide.

The invention also provides a method for producing protein peptide, which comprises the steps of inoculating the Streptomyces nigrolae (Streptomyces canus) T20 into a fermentation culture medium for fermentation to obtain a fermentation liquor containing proteolytic enzyme, extracting the fermentation liquor to obtain proteolytic enzyme, adding the proteolytic enzyme into a reaction system containing protein for enzymolysis to obtain an enzymolysis liquid containing protein peptide, and finally extracting the enzymolysis liquid to obtain the protein peptide;

or, the method comprises the steps of inoculating the Streptomyces nigrescens (Streptomyces canus) T20 into a fermentation medium for fermentation to obtain a fermentation liquid containing proteolytic enzyme, then adding the fermentation liquid into a reaction system containing protein for enzymolysis to obtain an enzymolysis liquid containing protein peptide, and finally extracting the enzymolysis liquid to obtain the protein peptide;

or, the method comprises the steps of inoculating the Streptomyces nigrospermi (Streptomyces canus) T20 into a fermentation culture medium for fermentation to obtain a fermentation liquid containing proteolytic enzyme, centrifuging the fermentation liquid to obtain a supernatant to obtain a fermentation supernatant, adding the fermentation supernatant into a reaction system containing protein for enzymolysis to obtain an enzymolysis liquid containing protein peptide, and finally extracting the enzymolysis liquid to obtain the protein peptide.

In one embodiment of the invention, the concentration of the protein in the reaction system is 5-50 g/L.

In one embodiment of the invention, the addition amount of the proteolytic enzyme, the fermentation liquid or the fermentation supernatant in the reaction system is 1000-3000U/g_Protein。

In one embodiment of the present invention, the temperature of the enzymolysis is 30 to 40 ℃, the rotation speed is 150 to 200rpm, and the pH is 7 to 8.

In one embodiment of the invention, the protein is rice protein; the protein peptide is rice peptide.

In one embodiment of the invention, the rice peptide is a small molecule rice peptide having a molecular weight of less than 1000 Da.

[ advantageous effects ]

(1) The invention screens out a Streptomyces nigrolae (Streptomyces canus) T20 which can efficiently degrade protein, and the Streptomyces nigrolae (Streptomyces canus) T20 is coated on a solid culture medium containing 1g/L rice protein to be cultured for 48h, so that the diameter of a transparent ring of the solid culture medium can reach 25 mm.

(2) The invention screens out a Streptomyces nigrolae (Streptomyces canus) T20, the Streptomyces nigrolae (Streptomyces canus) T20 can produce proteolytic enzyme with high-yield amino acid sequence as shown in SEQ ID NO.2, the Streptomyces nigrolae (Streptomyces canus) T20 is inoculated into a fermentation culture medium for fermentation for 48 hours, and the enzyme activity of the proteolytic enzyme in the fermentation supernatant can reach 1500U/mL.

(3) The invention provides a method for producing rice peptide, which greatly improves the content of small molecular peptide in the rice peptide by using fermentation supernatant obtained by fermenting Streptomyces griseus (Streptomyces canus) T20, and the fermentation supernatant obtained by fermenting the Streptomyces griseus (Streptomyces canus) T20 has the final enzyme activity of 2400U/g_ProteinThe additive amount of the small molecular peptides is added into rice protein dispersion liquid with the concentration of 10g/L of rice protein for enzymolysis, and the content of the small molecular peptides in enzymolysis supernatant can reach 70.3 percent only by enzymolysis for 2 hours.

(4) The invention provides a method for producing rice peptide, which only needs to add fermentation supernatant obtained by fermenting Streptomyces nigrescens (Streptomyces canus) T20 into rice protein dispersion liquid containing rice protein for enzymolysis, thus obtaining the rice peptide rich in small molecular peptide.

Biological material preservation

Streptomyces fuscous (Streptomyces canus) is classified and named as Streptomyces canus T20, is preserved in China Center for Type Culture Collection (CCTCC) at 11.15.2019, has a preservation number of M2019936, and has a preservation address of Wuhan university, Wuhan China.

Drawings

FIG. 1: phylogenetic tree of Streptomyces glaucescens (Streptomyces canus) T20.

FIG. 2: a colony of Streptomyces griseus (Streptomyces canus) T20.

Detailed Description

The invention is further illustrated with reference to specific examples.

The rice protein powder referred to in the following examples was purchased from Wuxi Jinnong Biotech limited, and other reagent materials were purchased from the national pharmaceutical group.

The media involved in the following examples are as follows:

enrichment culture medium: 1g/L of rice protein powder.

Separating a culture medium: 1g/L of rice protein powder, 3g/L of glucose and 15g/L of agar.

Fermentation medium: 2.5g/L rice protein powder, 0.1g/L sodium dihydrogen phosphate, 0.1g/L disodium hydrogen phosphate, 0.05g/L magnesium sulfate, 0.001g/L ferrous sulfate, and pH 7.0.

LB liquid medium: 10g/L of peptone and 5g/L, NaCl 10g/L of yeast extract.

LB solid medium: 10g/L of peptone, 5g/L, NaCl 10g/L of yeast extract and 15g/L of agar.

NaCl-tolerant medium: 0-10 g/L of NaCl, 10g/L of peptone and 5g/L of yeast extract.

The detection methods referred to in the following examples are as follows:

determination of proteolytic enzyme activity:

drawing a standard curve: weighing a certain amount of paranitroaniline, dissolving the paranitroaniline by using a small amount of absolute ethyl alcohol, and diluting the paranitroaniline by using a Tris-HCl buffer solution with the pH value of 8.0 until the final concentration is 40 mu g/mL; taking the diluted paranitroaniline solution as mother liquor, respectively taking 1000, 950, 900, 850, 800, 750, 700, 650, 600, 550, 500, 450, 400, 350, 300, 250, 200, 150, 100, 50 and 0 mu L of the mother liquor, supplementing the mother liquor to 1000mL by using Tris-HCl buffer solution with the concentration of 50mM and the pH value of 9.0, and preparing a series of standard solutions with concentration gradients (0.0, 2.0, 4.0, 6.0, 8.0, 10.0, 12.0, 14.0, 16.0, 18.0, 20.0, 22.0, 24.0, 26.0, 28.0, 30.0, 32.0, 34.0, 36.0, 38.0 and 40.0 mu g/mL); taking Tris-HCl buffer solution with the pH value of 8.0 as blank control, and measuring the absorbance of the standard solution at the wavelength of 405 nm; OD is determined by taking the concentration of paranitroaniline as the abscissa and the absorbance as the ordinate₄₀₅A linear graph of the concentration relation of the enzyme activity and the paranitroaniline is an enzyme activity standard curve;

and (3) measuring enzyme activity: adding 600 μ L of Tris-HCl buffer solution with the concentration of 50mM and the pH value of 8.0 and 50 μ L of solution to be detected into a 1.5mL EP tube; preheating the mixed solution in a water bath at 60 ℃ for 5min, adding 50 mu L of Leu-pNA solution with the concentration of 100mM into the mixed solution, and continuing to react in the water bath at 60 ℃ for 10 min; after the reaction is finished, completely placing the EP tube in ice to terminate the reaction, and measuring the light absorption value of the reaction solution under the wavelength of 405nm by using an enzyme-labeling instrument; calculating the enzyme activity of the proteolytic enzyme in the enzyme solution to be detected according to the light absorption value;

wherein, the calculation formula of the enzyme activity is as follows:

enzyme activity (U/mL) ═ OD₄₀₅×7×D/(K×10×0.5)；

The definition of enzyme activity is: the amount of the enzyme required to release 1. mu. mol of p-nitroanilide (p-nitroanilide) per minute at a pH of 8.0 and a temperature of 60 ℃ was one unit of the enzyme activity (1U).

Determination of rice peptide molecular weight distribution:

taking rice peptide as a sample, adding distilled water to dissolve the rice peptide into a solution with the concentration of 5mg/mL, centrifuging at 12000rpm for 10min, and taking 200 mu L of supernatant to perform gel chromatography analysis; the chromatographic conditions were as follows: TSKgel G2000 SW_XL(7.8 mm. times.30 cm, Tosoh); a diode array detector for detecting the wavelength of 220 nm; mobile phase: v (acetonitrile): v (water): v (trichloroacetic acid) ═ 10:90: 0.1; the column temperature is 30 ℃; sample introduction amount: 20 mu L of the solution; the flow rate is 0.8 mL/min; data analysis was performed using a V2.0 english spectral gel chromatography data workstation.

Example 1: primary screen of streptomyces glaucescens T20

The method comprises the following specific steps:

taking soil in a factory area of a flour mill from a shores and lakes area of Wuxi city in Jiangsu province as a sample, weighing 10g of the sample, adding the sample into 90mL of sterilized enrichment medium, and performing shake culture at 30 ℃ and 200rpm for 48 hours to obtain a culture solution; sucking 10mL of culture solution, adding the culture solution into a new 90mL of sterilized enrichment culture medium, performing shake culture at 30 ℃ and 200rpm for 48 hours, and repeating for 2 times to obtain an enrichment solution; diluting the enrichment solution with sterile water in a gradient manner to obtain a gradient dilution solution; respectively diluting 200 μ L to 10^-3、10^-4、10^-5、10^-6、10^-7、10^-8The gradient diluent is coated on a separation culture medium, cultured for 48 hours at the temperature of 30 ℃, and a single colony with a transparent ring is selected; selecting a single colony with a transparent ring, streaking the single colony on an LB solid culture medium, culturing the colony for 48 hours at the temperature of 30 ℃, and repeating the steps for many times until the single-form thallus can be observed under a microscope to obtain a purified single colony; the purified single colonyInoculating to separate culture medium, culturing at 30 deg.C for 48 hr, and selecting strain with larger transparent ring (diameter)>20mm) respectively named T16, T20, T37, T39, T40, T53 (see table 1 for transparent circle diameters of strains T16, T20, T37, T39, T40, T53).

TABLE 1 transparent circle diameter of strains T16, T20, T37, T39, T40, T53

Bacterial strains	Diameter of transparent ring (mm)
		T16	20
T20	25
		T37	20
T39	22
		T40	20
T53	21

Example 2: rescreening of streptomyces glaucescens T20

The method comprises the following specific steps:

the single colonies purified from the strains T16, T20, T37, T39, T40 and T53 obtained in example 1 were inoculated into LB liquid medium at 30 ℃ and 200rCulturing for 24 hours under pm shaking to obtain seed liquid; inoculating the seed solution into fermentation medium at an inoculum size of 5% (v/v), and shake-culturing at 30 deg.C and 200rpm for 48 hr to OD of the fermentation liquid₆₀₀1.2; centrifuging the fermentation liquor at 12000rpm and 4 deg.C for 20min to obtain fermentation supernatant; dissolving rice protein powder in distilled water according to a feed-liquid ratio of 10:1(w/v, g/L) to obtain a dispersion liquid; adding the fermentation supernatant into the dispersion to make the final enzyme activity of the proteolytic enzyme in the dispersion 2400U/g_ProteinReacting in a constant-temperature water bath shaking table at 40 ℃ and 150rpm for 2h, boiling for 15min, and inactivating enzyme to obtain enzymatic hydrolysate; centrifuging the enzymolysis solution at 5000rpm for 20min to obtain enzymolysis supernatant.

The enzymatic activity of the proteolytic enzyme in the fermentation supernatant was measured (see table 2 for the results of the measurements).

The dispersion added with the fermentation supernatant after 20min of boiling water bath treatment is used as a blank control, and the molecular weight distribution of the rice peptide in the filtrate is detected by gel chromatography analysis after the enzymolysis supernatant is filtered by a 0.45 mu M filter membrane (the detection result is shown in table 3).

As can be seen from Table 2, the highest enzyme activity of the proteolytic enzyme in the fermentation supernatant obtained by the fermentation of the strain T20 was 1500U/mL. Sequencing the obtained proteolytic enzyme, and finding that the amino acid sequence is shown as SEQ ID No.2 and the nucleotide sequence is shown as SEQ ID No. 3.

As can be seen from Table 3, the content of small-molecule peptides in the supernatant obtained by enzymolysis of the fermentation supernatant obtained by fermentation with the strain T20 is the highest, and accounts for 70.3% of the total protein content.

TABLE 2 enzymatic Activity of proteolytic enzymes in fermentation supernatants obtained by fermentation of strains T16, T20, T37, T39, T40, T53

Bacterial strains	Enzymatic Activity of proteolytic enzymes (U/mL)
		T16	940
T20	1500
		T37	906
T39	1230
		T40	920
T53	1106

TABLE 3 ratio of small molecular peptide and polypeptide content in total protein content in the fermented supernatant obtained by fermentation with strains T16, T20, T37, T39, T40 and T53

Strain numbering	Small peptide (< 1000Da,%)	Polypeptides (> 1000Da,%)
			Blank control	24.5	75.6
T16	41.2	56.2
			T20	70.3	29.19
T37	30.6	67.3
			T39	49.2	49.2
T40	51.6	47.1
			T53	60.3	38.4

Example 3: identification and preservation of Streptomyces griseus T20

The method comprises the following specific steps:

the strain T20 obtained in example 2 was selected, the total DNA of the strain T20 was extracted, 16SrDNA amplification and sequencing were performed (completed by Wutin Tianlin biotechnology, Inc., the nucleotide sequence of 16S rDNA of the strain T20 is shown in SEQ ID NO. 1), the sequence obtained by sequencing was subjected to nucleic acid sequence alignment in Genbank, it was found that the similarity of the 16S rDNA sequence thereof with the 16S rDNA sequence of Streptomyces griseus (Streptomyces canus) DSM 40017 was as high as 98.3%, the strain with the high similarity thereof was constructed as a phylogenetic tree (see FIG. 1 in particular), and the result showed that the strain and Streptomyces griseus (Streptomyces canus) cfcc 3169 belong to the same branch and was named as Streptomyces griseus (Streptomyces griseus) T20.

Sending Streptomyces griseus (Streptomyces canus) T20 to the China center for preservation with the preservation date as follows: 11 and 15 days in 2019, the preservation number is CCTCC NO: M2019936, and the preservation address is Wuhan, Wuhan university, China.

Example 4: culture and Observation of Streptomyces griseus T20

The method comprises the following specific steps:

a loop of purified single colonies of Streptomyces griseus (Streptomyces canus) T20 obtained in example 1 was scraped and streaked on LB solid medium, and after 24 hours of cultivation at 30 ℃, colonies were observed, and were found to be small, white, with regular edges, circular, and 2-4 mm in diameter (see FIG. 2 for details).

A loop of the purified single colony of Streptomyces griseus (Streptomyces canus) T20 obtained in example 1 was scraped and inoculated into an LB liquid medium with a pH of 7.0, and cultured at a constant temperature of 10-50 ℃ for 48 hours, respectively, and the growth temperature was found to be 20-45 ℃ and the optimum temperature was 30 ℃.

A loop of the purified single colony of Streptomyces griseus (Streptomyces canus) T20 obtained in example 1 was scraped out, inoculated into LB liquid medium with pH of 3.0-10.0, and incubated at 30 ℃ for 48 hours, and the growth pH was found to be 6.0-8.0, with the optimum pH being 7.0.

A single colony of purified Streptomyces griseus (Streptomyces canus) T20 obtained in example 1 was scraped out, inoculated into NCl-tolerant cultures each containing 0 to 10g/L of NCl, and incubated at a constant temperature of 30 ℃ at pH7.0 for 48 hours, and it was found that it can grow in the NCl-tolerant culture containing 0 to 5g/L of NCl, and that it grew most vigorously in the NCl-tolerant culture containing 1g/L of NCl.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Sequence listing

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ctggagggca cggcgaccct tctggcccgc accacgcagg acctgtcgcg gttcaacctg 300

gacttcctgc tcgatgtcag cgaggtgcgg gtcaacggcg tgaaggcggc gttcaccgcc 360

tccggggagc acgagctgga gatcacgccg aagaccccgc tggcgaaggg cgccgccgcg 420

acgatcgtcg tgcggtacag cggggtgccc tcgtcgaagc aggcgtacgg cttcaccagc 480

tggcaccgca ccccggacgg cggggtcggc gcgaacgagc ccgaggcggc ctggtggtgg 540

ttcccgagca acgatcatcc gctcgacaag gccacctacg acgtgtccgt actggttccg 600

gacggctccc aggccatctc caacggcacg ctccagtcga cgagttcacg ggcgggctgg 660

acccggttca actggcgctc caacaagccg caggccacct atctggcgac gctggccgtc 720

gggaagttcg acatcacgac cgggaggacc gagagcggca ttccggtcgt caacgcctac 780

agcaaggacc tcggggacaa cgcgggggcc gcgcgggcga gcatcgagcg gacaggagag 840

atcgccgact ggctgagcga gtactacggg ccgtacccgt acaacgcgct cggcgggtac 900

gtgccgaaca ccaacaccgg gtacgcgctg gagacgcaga cccggccgtt ctacagcccg 960

cggcagttcg cgggcgggtc caatgtgtcc gtggtcgtgc acgagctggc ccaccagtgg 1020

tacggcgacc tcgtctccgt cgccgggtgg aaggacatct ggatcaacga ggggttcgcg 1080

cggtacgccc agtggctgtg gtccgagcac gagaacgagg gcacggccca ggagatcgcg 1140

gactacgtgt acgcctcgca cccggccgac gacccgttct ggacggtgaa gcccggtgac 1200

cccgggccgg agaaccagtt ccacctcgcc gtctacgacc ggggcgggct ggccctgcag 1260

gcgctgcgca acgagatcgg ggacgacgac ttcttcgcga tcctgaaggg ctggccgcag 1320

aagtacgcgt acgggaacgc gacggtcgcc gacttcgaga aatacgccga ggaggtgtcc 1380

gggcagtcgc tgtcggcgct cttcgacacc tggctgttcc agccgtcgaa gccggccgcc 1440

ccggcggcgg cggacgcgtc catcgcgcgg gcggcggccg ccggggagac gccggtgcgg 1500

ccgaagtcgt ggaagaagat cgccgcgacg aatgacgtgc acgagcgctg a 1551

Claims

1. A Streptomyces glaucous (Streptomyces canus) T20 is characterized in that the Streptomyces glaucous T20 is preserved in China Center for Type Culture Collection (CCTCC) at 11 months and 15 days in 2019 with the preservation number of M2019936.

2. Use of the Streptomyces glaucous T20 according to claim 1 for the production of proteolytic enzymes.

3. A method for producing proteolytic enzyme, characterized in that, the method comprises the steps of inoculating the Streptomyces griseus T20 of claim 1 into a fermentation medium for fermentation to obtain a fermentation liquor containing proteolytic enzyme, and then extracting the fermentation liquor to obtain the proteolytic enzyme.

4. The method for producing a proteolytic enzyme according to claim 3, wherein the proteolytic enzyme has the amino acid sequence shown in SEQ ID No. 2.

5. The method for producing proteolytic enzyme according to claim 3 or 4, wherein the fermentation temperature is 20 to 45 ℃, the rotation speed is 100 to 200rpm, and the pH is 6.0 to 8.0.

6. Use of the Streptomyces glaucescens T20 according to claim 1 for the production of protein peptides.

7. A method for producing protein peptide, characterized in that, the method is to inoculate the Streptomyces griseus T20 of claim 1 into a fermentation medium for fermentation to obtain a fermentation broth containing proteolytic enzyme, then extract the fermentation broth to obtain proteolytic enzyme, then add the proteolytic enzyme into a reaction system containing protein for enzymolysis to obtain an enzymolysis solution containing protein peptide, and finally extract the enzymolysis solution to obtain protein peptide;

or, the method is that the streptomyces glaucescens T20 of claim 1 is inoculated in a fermentation medium for fermentation to obtain a fermentation liquid containing proteolytic enzyme, then the fermentation liquid is added into a reaction system containing protein for enzymolysis to obtain an enzymolysis liquid containing protein peptide, and finally the enzymolysis liquid is extracted to obtain the protein peptide;

or, the method is that the streptomyces glaucescens T20 of claim 1 is firstly inoculated in a fermentation culture medium for fermentation to obtain a fermentation liquid containing proteolytic enzyme, then the fermentation liquid is centrifuged to obtain supernatant fluid, fermentation supernatant fluid is obtained, then the fermentation supernatant fluid is added into a reaction system containing protein for enzymolysis to obtain enzymolysis liquid containing protein peptide, and finally the enzymolysis liquid is extracted to obtain the protein peptide.

8. The method for producing protein peptide according to claim 7, wherein the temperature of the enzymolysis is 30-40 ℃, the rotation speed is 150-200 rpm, and the pH is 7-8.

9. The method for producing a protein peptide according to claim 7 or 8, wherein the protein is rice protein; the protein peptide is rice peptide.

10. A method of producing protein peptides as claimed in claim 9, wherein the rice peptides have a molecular weight of less than 1000 Da.