CN102559537B

CN102559537B - Bacillus and application of bacillus in protease production

Info

Publication number: CN102559537B
Application number: CN 201110362012
Authority: CN
Inventors: 温廷益; 邓爱华; 吴杰
Original assignee: Institute of Microbiology of CAS
Current assignee: Institute of Microbiology of CAS
Priority date: 2011-11-15
Filing date: 2011-11-15
Publication date: 2013-06-12
Anticipated expiration: 2031-11-15
Also published as: CN102559537A

Abstract

The invention discloses a bacillus and application of the bacillus in protease production. In the invention, the preservation number of the provided bacillus (Bacillus sp.) BS036 for producing protease is CGMCC (China General Microbiological Culture Collection Center) NO.5178. The bacillus for producing protease is high in protease output, simple in culture medium fermentation formula, wide in source of cheap raw materials and low in cost. The invention also provides a method for efficiently producing protease by using the bacillus; and the method is adopted to shorten the production cycle of the protease, the protease production is stable, the cost is low and the protease output can reach1000-100000U/ml.

Description

One bacillus and the application in producing proteolytic enzyme thereof

Technical field

The present invention relates to a bacillus and the application in producing proteolytic enzyme thereof.

Background technology

Proteolytic enzyme (Protease) can the protein hydrolysate peptide bond, extensively is present in bacterium, actinomycetes, fungi, virus, Plants and Animals body, plays a significant role in nature material cycle and organism metabolism.Proteolytic enzyme not only has vital role in nature and organism, and is a kind of very important industrial biological catalyst, adopts zymin to replace the traditional chemical production technique, plays an important role at aspects such as reducing pollution, minimizing energy consumption, protection of the environment.Since the fifties in 19th century zymin found first, be widely used in various production industries, as fields such as washing composition, food, makeup, aquatic feeds, process hides, weaving, medicine and biological chemical reagents, accounted for 60% of world's zymin total sales volume at present.

Along with fast development and the economic swift and violent lifting of society, every profession and trade is more and more higher to kind and the performance requriements of zymin, requires again the zymin cost further to reduce simultaneously.Industrial protease deep fermentation method commonly used is produced at present, also can produce with solid state fermentation, wherein substratum has occupied the production cost of 30%-40%, thereby after obtaining to be applicable to industrial bacterium producing multi enzyme preparation, also must be optimized to obtain best production technique to the fermentation condition of bacterial classification.Because proteolytic enzyme generally produces in a large number in growth stationary phase, its fermentative production is subject to the regulation and control of catabolic repression, and production of enzyme also is subject to the impact of carbon nitrogen to a great extent.In addition, other chemical factors also can affect production of enzyme as factors such as dissolved oxygen amount, inoculum size, culture temperature and medium pHs, is difficult to realize that the efficient stable of proteolytic enzyme output improves.Therefore, adopting fermentation condition optimization can realize the efficient stable production of proteolytic enzyme, is one of important method that reduces proteolytic enzyme industrial production cost, and the proteolytic enzyme suitability for industrialized production is had very important realistic meaning.

Although present above-mentioned proteolytic enzyme fermentation technique can improve the proteolytic enzyme fermentation production efficiency to a certain extent, but also there is following problem and shortage part: one, most bacterial strain production of enzyme are not high, after fermentation condition optimization, production of enzyme improves not significantly, often produces in actual applications undesirable result; They are two years old, substratum that technology is used often is the compound nutritional composition of high density mostly, and fermentation raw material is more expensive, although also there are some technology also to adopt cheap raw material, but production of enzyme does not obviously improve, thereby does not obviously reduce production costs on the fermentation raw material of unit production of enzyme.

Summary of the invention

The purpose of this invention is to provide a bacillus and the application in producing proteolytic enzyme thereof.

The invention provides a strain and produce genus bacillus (Bacillus sp.) BS036 of proteolytic enzyme, this bacterial classification has been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (abbreviation CGMCC on 08 26th, 2011, the address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City), preserving number is CGMCC No.5178.Genus bacillus (Bacillus sp.) BS036 CGMCC No.5178 is called for short bacterial strain BS036.Genus bacillus claims again bacillus.

The present invention also protects a kind of seed culture medium, is comprised of solute and water; Described solute and the concentration in described seed culture medium thereof are as follows: glucose 4-20g/L (being preferably 5-15g/L), peptone 3-8g/L, yeast powder 3-8g/L, KH ₂PO ₄0.5-1.5g/L, MgSO ₄0.1-1.0g/L and Na ₂CO ₃5-15g/L.Described seed culture medium can be nature pH, can be also pH7.0-11.5, is preferably pH11.0.Described solute and the concentration in described seed culture medium thereof are specific as follows: glucose 10g/L, peptone 5g/L, yeast powder 5g/L, KH ₂PO ₄1g/L, MgSO ₄0.2-0.8g/L and Na ₂CO ₃10g/L.

The present invention also protects a kind of fermention medium, is comprised of solute and water; Described solute is comprised of carbon source, nitrogenous source and inorganic salt; Described carbon source can be at least a in glucose, Semen Maydis powder, Zulkovsky starch, molasses, maltose and Testa Tritici; Described nitrogenous source can be at least a in cottonseed meal, soybean cake powder, soy peptone, casein, water-soluble plant protolysate, fish peptone, yeast powder and Oxide peptone; Described inorganic salt can be Trisodium Citrate, NaNO ₃, CaCl ₂, FeSO ₄, CuSO ₄, ZnCl ₂, KH ₂PO ₄, MgSO ₄And Na ₂CO ₃In at least a.

Described fermention medium can be nature pH, can be also pH7.0 to 11.5 (7.0,8.0,9.0,9.5,10.0,10.5,11.0 or 11.5), is preferably pH9.0-11.0, most preferably is pH11.0.

In every liter of described fermention medium, the add-on of described carbon source can be 4-24g.

In every liter of described fermention medium, the add-on of described nitrogenous source can be 4-20gL.

In every liter of described fermention medium, the add-on of every kind of described inorganic salt can be 0.064-20g.

described carbon source can be glucose, and (every liter of fermention medium adds 4, 8, 12, 16 or 20g), (every liter of fermention medium adds 6 to Semen Maydis powder, 9, 12, 15, 18, 21 or 24g), (every liter of fermention medium adds 6 to Zulkovsky starch, 9, 12, 15 or 18g), (every liter of fermention medium adds 6 to molasses, 9, 12, 15, 18 or 21g), (every liter of fermention medium adds 6 to maltose, 9, 12, 15 or 18g) and Testa Tritici (every liter of fermention medium adds 6, 9, 12, 15, 18 or 21g) at least a.

described nitrogenous source can be cottonseed meal, and (every liter of fermention medium adds 6, 8, 10, 12 or 14g), (every liter of fermention medium adds 4 to soybean cake powder, 6, 8, 10, 12 or 14g), (every liter of fermention medium adds 8 to soy peptone, 10, 12, 14 or 16g), (every liter of fermention medium adds 8 to casein, 10, 12, 14 or 16g), (every liter of fermention medium adds 1 to water-soluble plant protolysate, 2, 3, 4, 5, 6 or 7g), (every liter of fermention medium adds 8 to fish peptone, 10, 12, 14 or 16g), (every liter of fermention medium adds 8 to yeast powder, 10, 12, 14 or 16g) and the Oxide peptone (every liter of fermention medium adds 6, 8, 10, 12, 14, 16, 18 or 20g) at least a.

Described inorganic salt can be Trisodium Citrate (every liter of fermention medium add 2,3,4,5 or 6g), NaNO ₃(every liter of fermention medium add 4,8,12,16 or 20g), CaCl ₂(every liter of fermention medium add 0.2,0.4,0.6,0.8 or 1.0g), FeSO ₄7H ₂O (every liter of fermention medium add 0.6,1.2,1.8,2.4 or 3.0g), CuSO ₄5H ₂O (every liter of fermention medium add 0.1,0.2,0.3,0.4 or 0.5g), ZnCl2 (every liter of fermention medium add 0.1,0.2,0.3,0.4 or 0.5g), KH ₂PO ₄(every liter of fermention medium add 0.4,0.8,1.2,1.6 or 2.0g), MgSO ₄(every liter of fermention medium add 0.2,0.4,0.6,0.8 or 1.0g) and Na ₂CO ₃At least a in (every liter of fermention medium add 5.0,10.0 or 15.0g).

Described solute and the concentration in described fermention medium thereof are as follows: glucose 5-15g/L, peptone 3-8g/L, yeast powder 3-8g/L, KH ₂PO ₄0.5-1.5g/L, MgSO ₄0.1-1.0g/L and Na ₂CO ₃5-15g/L.Described solute and the concentration in described fermention medium thereof are specific as follows: glucose 10g/L, peptone 5g/L, yeast powder 5g/L, KH ₂PO ₄1g/L, MgSO ₄0.2-0.8g/L and Na ₂CO ₃10g/L.

Described solute and the concentration in described fermention medium thereof are as follows: Semen Maydis powder 18.0-20.0g/L (18.0g/L, 18.9g/L, 19.0g/L or 20.0g/L), soy peptone 6.0-7.0g/L (6.0g/L, 6.1g/L, 6.5g/L or 7.0g/L), Trisodium Citrate 2.0-6.0g/L (2.0g/L, 4.0g/L or 6.0g/L), MgSO ₄(0.1-1.2g/L 0.1g/L, 0.4g/L, 0.7g/L or 1.2g/L), KH ₂PO ₄(1.0-1.2g/L 1.0g/L, 1.1g/L or 1.2g/L), CaCl ₂(0.2-1.2g/L 0.2g/L, 1.0g/L or 1.2g/L) and Na ₂CO ₃10.0g/L.

The present invention also protects a kind of method for preparing the seed liquor of described genus bacillus BS036, comprises the steps: described genus bacillus BS036 is inoculated in described seed culture medium, and 26-37 ℃, 120-220rpm shaking culture obtain seed liquor.Described vibration is preferably the 200rpm vibration.The temperature of described shaking culture is preferably 30-34 ℃, most preferably is 34 ℃.The OD of described seed liquor ₆₀₀For 0.5-18.0 (0.5,1.0,3.0,7.0,9.0,12.0,14.0 or 18.0), be preferably 3.0-12.0, more preferably 7.0 to 12.0.

The present invention also protects a kind of method for preparing proteolytic enzyme, is the described genus bacillus BS036 of fermentation, obtains proteolytic enzyme.Described method comprises the steps: the seed liquor that the above method prepares is inoculated in described fermention medium, and 26-37 ℃, 120-220rpm shaking culture obtain proteolytic enzyme.Described vibration is preferably the 200rpm vibration.The temperature of described shaking culture can be 26-37 ℃ (26 ℃, 28 ℃, 30 ℃, 32 ℃, 34 ℃ or 37 ℃), is preferably 30-37 ℃, most preferably is 34 ℃.The inoculum size of described seed liquor can be 0.5%-5.0% (0.5%, 1.0%, 2.0%, 3.0%, 4.0% or 5.0%), is preferably 1.5%-5.0%, most preferably is 2.0%.The time of described shaking culture can be 3-48 hour, is preferably 24-40 hour, most preferably is 27-30 hour.Described method also can comprise the steps: the centrifugal 10min of fermented liquid 12000 * g that described shaking culture is obtained, collects supernatant, is crude enzyme liquid (containing proteolytic enzyme).

The proteolytic enzyme that the above method prepares also belongs to protection scope of the present invention.

The present invention also protects the application of proteolytic enzyme in protein degradation of adopting the above method to prepare.When using the described albumen of described proteasome degradation, the temperature of employing is 20-90 ℃, and the pH of employing is 5.0-13.0.Described temperature can be 20-90 ℃ (20 ℃, 30 ℃, 40 ℃, 50 ℃, 55 ℃, 60 ℃, 65 ℃, 70 ℃, 80 ℃ or 90 ℃), is preferably 60-65 ℃.Described pH can be 5.0-13.0 (5.0,6.0,7.0,8.0,9.0,10.0,11.0,12.0 or 13.0), is preferably 10.0-11.0.Described albumen specifically can be casein or coupling casein.

The application of described genus bacillus BS036 in producing proteolytic enzyme also belongs to protection scope of the present invention.

The invention provides a strain good bacterial strain of white enzyme performance of laying eggs.Protease production strain kind production of enzyme of the present invention is high, and the fermentative production culture medium prescription is simple, and cheap raw material wide material sources, price are low, and fermentation period is short, consumes energy low, and proteolytic enzyme production efficiency is high, cost is low.The present invention also provides the method for using described bacterial strain High-efficient Production proteolytic enzyme, and the production cycle of employing this method enzyme shortens, the product enzyme is stable, cost is low, and production of enzyme can reach 1000-100000U/mL.

Description of drawings

Fig. 1 is ITS and the GyrB gene product electrophorogram of pcr amplification bacterial classification BS036.

Fig. 2 is substratum factor interphase interaction three-dimensional space response surface chart and the corresponding two-dimentional isogram of bacillus protein enzymic fermentation: A1 and A2 are the interaction diagram of Semen Maydis powder and soy peptone; B1 and B2 are Semen Maydis powder and KH ₂PO ₄Interaction diagram; C1 and C2 are soy peptone and KH ₂PO ₄Interaction diagram.

Fig. 3 is that polyacrylamide gel electrophoresis detects the genus bacillus BS036 different fermentations extracellular protease in period.

Fig. 4 is the leaven line chart that utilizes genus bacillus BS036 fermentation production of protein enzyme.

Embodiment

Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique in following embodiment if no special instructions, is ordinary method.Test materials used in following embodiment if no special instructions, is and purchases available from routine biochemistry reagent shop.Quantitative test in following examples all arranges repeated experiments three times, results averaged.Coupling casein (Azocasein): available from U.S. Sigma-Aldrich company, article No. is A2765.Inoculum size (%) is the volume ratio of seed liquor and fermention medium.

Proteinase activity method for measuring: take coupling casein (Azocasein) as substrate, substrate is dissolved in the substrate buffer solution that is made into 1g/100mL in damping fluid; Get the preheating 5 minutes in the water-bath of preset temperature of 1.49mL substrate buffer solution, then add 10 μ L solution to be measured, short mix, reaction is 5 minutes under described preset temperature, then add 0.5mL 20% (volume ratio) trichloroacetic acid solution termination reaction, the centrifugal 10min of 12000 * g collects supernatant, measures the absorbance value of supernatant liquor under the 440nm wavelength; An enzyme activity unit (U) is defined as the per minute absorbance value and changes 0.001 needed enzyme amount.

The screening of embodiment 1, bacterial strain BS036 and evaluation

One, the screening of bacterial strain BS036

Utilize the rich protein enzyme-producing bacteria strain from fertilizer heap sample of 1% casein solution, cultured continuously in the casein substratum is at the maximum bacterial strain of casein dull and stereotyped top sieve sortilin hydrolysis circle, called after bacterial strain BS036.

Two, the plysiochemical characterized of bacterial strain BS036

Bacterial strain BS036 was cultivated on solid medium 8-24 hour, and bacterium colony is yellow spheroidal, moistening thickness, opaque, and bacterium colony becomes large gradually with incubation time.By gramstaining and microscopic examination, this bacterium is shaft-like, Gram-positive.Physiology and biochemistry experiment shows, bacterial strain BS036 can grow under 15-40 ℃, the condition of pH 7-11, has the ability of protolysate and starch, and concrete physiological and biochemical property sees Table 1.

The physiological and biochemical property of table 1 bacterial strain BS036

Physiological and biochemical property	Result (+positive/-feminine gender)
		Shape	Shaft-like

Gramstaining	+
		The hydrolysis urobenzoic acid	-
The hydrolysis polysorbas20	+
		The hydrolysis polysorbate40	+
The hydrolysis polysorbate60	+
		The hydrolysis tween 80	-
Hydrolyzed casein	+
		Gelatin hydrolysate	-
Hydrolyzed starch	+
		Catalase activity	+
H ₂The S test	-
		Produce indole test	+
Methyl red test	-
		Fu-Pu test	-
Phenylalanine deaminase	-

Three, the genetics characteristics of bacterial strain BS036 is identified

Extract the chromosomal DNA of bacterial strain BS036, pcr amplification is also measured 16S rDNA sequence, by the 16S rDNA sequence similarity the highest (having 99% similarity) of sequence alignment and known genus bacillus.For further determining respectively its ITS and gyrB gene order to be increased the molecular genetic characteristic of this bacterial strain and analyzed.

Universal primer 16S-F-MYC (GGTGAATACGTTCTCGGGTCTTGTACACAC) and 23S-R1-MYC (TNCTTTTCACCTTTCCCTCACGGTAC) according to bacterium ITS sequence, the ITS sequence (seeing Figure 1A) of pcr amplification bacterial strain BS036, further order-checking obtains big or small be the DNA sequence dna of 1109bp (seeing the sequence 1 of sequence table).BLAST software by the NCBI website carries out homology with the ITS sequence of bacterial strain BS036 and known array at present to be compared, with the ITS sequence similarity the highest (having 90% similarity) of Bacillus clausii KSM-K16.

Universal primer BS-F (GAAGGCGGNACNCAYGAAG) and BS-R (CTTCRTGNGTNCCGCCTTC) according to bacterium GyrB, the GyrB portion gene sequence (seeing Figure 1B) of pcr amplification bacterial strain BS036, order-checking obtain size and are the DNA sequence dna of 1257bp (seeing the sequence 2 of sequence table).BLAST software by the NCBI website carries out homology with the DNA sequence dna of the DNA helicase subunit B of bacterial strain BS036 and known array at present to be compared, with the GyrB gene order similarity the highest (having 75% similarity) of Bacillus megaterium QM B1551.The sequence 3 that the Partial Protein sequence of inferring according to the GyrB portion gene sequence of bacterial strain BS036 is seen sequence table, BLAST software by the NCBI website carries out homology with the protein sequence of the DNA helicase subunit B of bacterial strain BS036 and known array at present to be compared, with the GyrB protein similar the highest (having 89% similarity) of Bacillus clausii KSM-K16.

According to form, physiological and biochemical property and genetics characteristics, bacterial strain BS036 is accredited as genus bacillus (Bacillus sp.).Genus bacillus (Bacillus sp.) BS036, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (abbreviation CGMCC on 08 26th, 2011, the address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City), preserving number is CGMCC No.5178.Genus bacillus (Bacillus sp.) BS036 CGMCC No.5178 is called for short bacterial strain BS036.

The mensuration of embodiment 2, protease hydrolysis performance

Seed culture medium is comprised of solute and water, and described solute and the concentration in described seed culture medium thereof are as follows: glucose 10g/L, peptone 5g/L, yeast powder 5g/L, KH ₂PO ₄1g/L, MgSO ₄0.2g/L and Na ₂CO ₃10g/L.

The formula of fermention medium is consistent with seed culture medium.

One, the preparation of proteolytic enzyme crude enzyme liquid

1, bacterial strain BS036 is inoculated in seed culture medium (natural pH), 30 ℃, 200rpm shaking culture are to bacterium liquid OD ₆₀₀Reach 7.0, obtain seed liquor.

2, the 1mL seed liquor is seeded in 50mL fermention medium (natural pH), 30 ℃, 200rpm shaking culture 30 hours are collected fermented liquid.

3, with the centrifugal 10min of fermented liquid 12000 * g, collect supernatant, be crude enzyme liquid (containing proteolytic enzyme).

Two, crude enzyme liquid is as the optimum reaction conditions of proteolytic enzyme

1, crude enzyme liquid is as the optimal reactive temperature of proteolytic enzyme

Detecting crude enzyme liquid lives as the enzyme of proteolytic enzyme.

Adopt respectively following preset temperature: 20 ℃, 30 ℃, 40 ℃, 50 ℃, 55 ℃, 60 ℃, 65 ℃, 70 ℃, 80 ℃, 90 ℃.

Adopt the 50mmol/L Tris-HCl damping fluid of following damping fluid: pH8.0.

When temperature of reaction was 20 ℃, the enzyme activity of 1mL crude enzyme liquid was 402.22 ± 94.36U.

When temperature of reaction was 30 ℃, the enzyme activity of 1mL crude enzyme liquid was 2015.56 ± 85.98U.

When temperature of reaction was 40 ℃, the enzyme activity of 1mL crude enzyme liquid was 5497.78 ± 165.64U.

When temperature of reaction was 50 ℃, the enzyme activity of 1mL crude enzyme liquid was 12968.89 ± 610.48U.

When temperature of reaction was 55 ℃, the enzyme activity of 1mL crude enzyme liquid was 13853.33 ± 205.26U.

When temperature of reaction was 60 ℃, the enzyme activity of 1mL crude enzyme liquid was 22320.00 ± 736.24U.

When temperature of reaction was 65 ℃, the enzyme activity of 1mL crude enzyme liquid was 21577.78 ± 159.07U.

When temperature of reaction was 70 ℃, the enzyme activity of 1mL crude enzyme liquid was 16733.33 ± 516.57U.

When temperature of reaction was 80 ℃, the enzyme activity of 1mL crude enzyme liquid was 8751.11 ± 688.78U.

When temperature of reaction was 90 ℃, the enzyme activity of 1mL crude enzyme liquid was 5637.78 ± 147.92U.

Crude enzyme liquid all has protease activity under 20-90 ℃ of reaction conditions, the enzymic activity under 60-65 ℃ of reaction conditions is the highest, and namely crude enzyme liquid is 60-65 ℃ as the optimal reactive temperature of proteolytic enzyme.

2, crude enzyme liquid is as the optimal reaction pH of proteolytic enzyme

Detecting crude enzyme liquid lives as the enzyme of proteolytic enzyme.

Adopt following preset temperature: 60 ℃.

Adopt respectively 50mmol/L Sodium phosphate dibasic-phosphate sodium dihydrogen buffer solution of following damping fluid: pH5.0, pH6.0 and pH7.0; The 50mmol/L Tris-HCl damping fluid of pH8.0 and pH9.0; 50mmol/L glycine-sodium hydrate buffer solution of pH10.0 and pH 11.0; Sodium carbonate-sodium hydrate buffer solution of pH12.0 and pH13.0.

Reaction pH is 5.0 o'clock, and the enzyme activity of 1mL crude enzyme liquid is 4641.00 ± 873.21U.

Reaction pH is 6.0 o'clock, and the enzyme activity of 1mL crude enzyme liquid is 5922.00 ± 1019.31U.

Reaction pH is 7.0 o'clock, and the enzyme activity of 1mL crude enzyme liquid is 10633.00 ± 1560.98U.

Reaction pH is 8.0 o'clock, and the enzyme activity of 1mL crude enzyme liquid is 22225.00 ± 5422.41U.

Reaction pH is 9.0 o'clock, and the enzyme activity of 1mL crude enzyme liquid is 22162.00 ± 4590.19U.

Reaction pH is 10.0 o'clock, and the enzyme activity of 1mL crude enzyme liquid is 24948.00 ± 6547.59U.

Reaction pH is 11.0 o'clock, and the enzyme activity of 1mL crude enzyme liquid is 25690.00 ± 8463.22U.

Reaction pH is 12.0 o'clock, and the enzyme activity of 1mL crude enzyme liquid is 13752.67 ± 350.37U.

Reaction pH is 13.0 o'clock, and the enzyme activity of 1mL crude enzyme liquid is 8372.00 ± 606.22U.

Crude enzyme liquid all has protease activity under the pH5.0-13.0 reaction conditions, the enzymic activity under the pH10.0-11.0 reaction conditions is the highest, and namely crude enzyme liquid is 10.0-11.0 as the optimal reaction pH of proteolytic enzyme.

Embodiment 3, bacterial strain BS036 produce the optimization of the suitableeest fermentation parameter of proteolytic enzyme

One, the optimization of the most adaptable method

The formula of seed culture medium and fermention medium is with embodiment 2.

1, optimize the suitableeest kind of age

(1) bacterial strain BS036 is inoculated in seed culture medium (natural pH), 30 ℃, 200rpm shaking culture obtain seed liquor.Get respectively the seed of different growing stage, i.e. the OD of seed liquor ₆₀₀Be respectively 0.5,1.0,3.0,9.0,12.0,14.0 and 18.0.

(2) respectively the 1ml seed liquor is seeded in 50ml fermention medium (natural pH), 30 ℃, 200rpm shaking culture 24 hours are collected fermented liquid.

(3) with the centrifugal 10min of fermented liquid 12000 * g, collect supernatant, be crude enzyme liquid.

(4) detecting crude enzyme liquid lives as the enzyme of proteolytic enzyme and (adopts 60 ℃ as preset temperature: the 50mmol/L glycine-sodium hydrate buffer solution that adopts following damping fluid: pH10.0).

The results are shown in Table 2.

Production of enzyme under table 2 fermentation conditions in age not of the same race

Each kind fermentation in age obtains crude enzyme liquid	The enzyme activity of 1mL crude enzyme liquid (U)
		Adopt OD ₆₀₀Value is the crude enzyme liquid that 0.5 seed liquor obtains by above-mentioned steps	17808.00±1107.91
Adopt OD ₆₀₀Value is the crude enzyme liquid that 1.0 seed liquor obtain by above-mentioned steps	20032.00±123.16
		Adopt OD ₆₀₀Value is the crude enzyme liquid that 3.0 seed liquor obtain by above-mentioned steps	23344.00±569.13
Adopt OD ₆₀₀Value is the crude enzyme liquid that 9.0 seed liquor obtain by above-mentioned steps	27128.00±390.20

Adopt OD ₆₀₀Value is the crude enzyme liquid that 12.0 seed liquor obtain by above-mentioned steps	31840.00±521.60
		Adopt OD ₆₀₀Value is the crude enzyme liquid that 14.0 seed liquor obtain by above-mentioned steps	19136.00±186.42
Adopt OD ₆₀₀Value is the crude enzyme liquid that 18.0 seed liquor obtain by above-mentioned steps	12920.00±1912.54

OD ₆₀₀Value is for seed liquor (namely reach the logarithmic growth seed liquor in mid-term, usually cultivated 8-12 hour) the inoculation fermentation substratum of 3.0-12.0 and after carrying out fermentation culture, and the enzyme of every milliliter of crude enzyme liquid is lived higher, and namely the output of proteolytic enzyme is higher.

2, inoculum size optimization

(1) bacterial strain BS036 is inoculated in seed culture medium (natural pH), 30 ℃, 200rpm shaking culture are to bacterium liquid OD ₆₀₀Reach 7.0, obtain seed liquor.

Seed liquor is seeded in fermention medium (natural pH) (2) that (following inoculum size is set respectively: 0.5%, 1.0%, 2.0%, 3.0%, 4.0%, 5.0%), 30 ℃, 200rpm shaking culture 24 hours are collected each fermented liquid.

(3) with the centrifugal 10min of each fermented liquid 12000 * g, collect supernatant, be each crude enzyme liquid.

The results are shown in Table 3.

Production of enzyme under table 3 different vaccination amount fermentation conditions

The crude enzyme liquid that different vaccination amount fermentative production obtains	The enzyme activity of 1mL crude enzyme liquid (U)
		The crude enzyme liquid that obtains by above-mentioned steps when the seed liquor inoculum size is 0.5%	20440.00±1360.74
The crude enzyme liquid that obtains by above-mentioned steps when the seed liquor inoculum size is 1.0%	25400.00±1386.94
		The crude enzyme liquid that obtains by above-mentioned steps when the seed liquor inoculum size is 2.0%	29440.00±1281.72
The crude enzyme liquid that obtains by above-mentioned steps when the seed liquor inoculum size is 3.0%	24720.00±895.99
		The crude enzyme liquid that obtains by above-mentioned steps when the seed liquor inoculum size is 4.0%	24560.00±576.54
The crude enzyme liquid that obtains by above-mentioned steps when the seed liquor inoculum size is 5.0%	24500.00±1126.23

Seed liquor is with the inoculum size inoculation fermentation substratum of 1.0%-5.0% and after carrying out fermentation culture, and the protease activity of crude enzyme liquid is all higher, and namely the output of proteolytic enzyme is all very high, and wherein production of enzyme is the highest when inoculum size is 2.0%.

3, leavening temperature optimization

(1) bacterial strain BS036 is inoculated in seed culture medium (natural pH), 30 ℃, 200rpm shaking culture are to the OD of bacterium liquid ₆₀₀Value reaches 7.0, obtains seed liquor.

(2) the 1mL seed liquor is seeded in 50mL fermention medium (natural pH), under 26 ℃, 28 ℃, 30 ℃, 32 ℃, 34 ℃, 37 ℃ equitemperature conditions, 200rpm shaking culture 24 hours is collected fermented liquid respectively.

The results are shown in Table 4.

Production of enzyme under the different leavening temperature condition of table 4

The crude enzyme liquid that different leavening temperatures obtains

The enzyme activity of 1mL crude enzyme liquid (U)

The crude enzyme liquid that adopts the leavening temperature of 26 ℃ to obtain	8484.67±464.79
		The crude enzyme liquid that adopts the leavening temperature of 28 ℃ to obtain	10794.67±342.48
The crude enzyme liquid that adopts the leavening temperature of 30 ℃ to obtain	23510.67±2302.06
		The crude enzyme liquid that adopts the leavening temperature of 32 ℃ to obtain	23826.00±6231.35
The crude enzyme liquid that adopts the leavening temperature of 34 ℃ to obtain	27368.00±2951.94
		The crude enzyme liquid that adopts the leavening temperature of 37 ℃ to obtain	20885.33±632.03

Under 30-37 ℃ of leavening temperature, the enzyme of crude enzyme liquid is lived all very high, and namely the output of proteolytic enzyme is all very high, and production of enzyme is the highest under the leavening temperature of 34 ℃.

4, optimum pH optimization

(2) fermention medium that the 1mL seed liquor is seeded to the various pH values of 50mL (is used Na ₂CO ₃Or NaOH regulates the pH to 7.0,8.0,9.0,9.5,10.0,10.5,11.0 or 11.5 of fermention medium), 30 ℃, 200rpm shaking culture 24 hours are collected fermented liquid.

The results are shown in Table 5.

Production of enzyme under the different fermentation pH value condition of table 5

The crude enzyme liquid that different fermentation pH obtains	The enzyme activity of 1mL crude enzyme liquid (U)
		The crude enzyme liquid that the fermentation pH of employing pH7.0 obtains	533.33±75.72
The crude enzyme liquid that the fermentation pH of employing pH8.0 obtains	1093.33±170.10
		The crude enzyme liquid that the fermentation pH of employing pH9.0 obtains	22933.33±1803.70
The crude enzyme liquid that the fermentation pH of employing pH9.5 obtains	25500.00±3000.00
		The crude enzyme liquid that the fermentation pH of employing pH10.0 obtains	28733.33±5937.45
The crude enzyme liquid that the fermentation pH of employing pH10.5 obtains	30917.33±3640.64
		The crude enzyme liquid that the fermentation pH of employing pH11.0 obtains	30960.00±2469.55
The crude enzyme liquid that the fermentation pH of employing pH11.5 obtains	10496.00±440.15

Under the fermentation condition of pH9.0-11.0, the enzyme of every milliliter of crude enzyme liquid is lived higher, and namely the output of proteolytic enzyme is higher.Production of enzyme is the highest under the fermentation condition of pH11.0.

Two, the optimization of suitable cheap carbon source

The formula of seed culture medium is with embodiment 2.

Fermention medium adopts different carbon sources or does not add carbon source, and every kind of carbon source arranges different concentration (0-24g/L concentration range), and other solute and content are with the fermention medium in embodiment 2 (in embodiment 2, the carbon source of fermention medium is glucose).Adopt respectively that process for processing in the market is simple, wide material sources, glucose, Semen Maydis powder, Zulkovsky starch, molasses, maltose, Testa Tritici (Testa Tritici) that price is low are carbon source.

Adopt respectively every kind of fermention medium to carry out following experimental procedure:

(1) bacterial strain BS036 is inoculated in seed culture medium (pH11.0), 34 ℃, 200rpm shaking culture are to the OD of bacterium liquid ₆₀₀Value reaches 7.0, obtains seed liquor.

(2) the 1mL seed liquor is seeded in 50mL fermention medium (pH11.0), 34 ℃, 200rpm shaking culture 24 hours are collected fermented liquid, obtain seed liquor.

The results are shown in Table 6.

The production of enzyme that table 6 different concns carbon source through fermentation obtains

During take Semen Maydis powder, molasses, glucose and maltose as carbon source, the proteolytic enzyme output of crude enzyme liquid is higher.

Three, the optimization of suitable cheap nitrogenous source

The formula of seed culture medium is with embodiment 2.

Fermention medium adopts different nitrogenous sources, and every kind of nitrogenous source arranges different concentration (0-20g/L concentration range), and other solute and content are with the fermention medium in embodiment 2 (in embodiment 2, the nitrogenous source of fermention medium is peptone and yeast powder).Adopt respectively import peptone and yeast powder in the alternative traditional zymotic substratum such as price is low in the market cottonseed meal, soybean cake powder, soy peptone, casein, water-soluble plant (wheat) protolysate, fish peptone, and with the yeast powder of different concns and Oxide peptone as reference.

Adopt respectively every kind of fermention medium to carry out the experimental procedure of step 2.

The results are shown in Table 7.

The production of enzyme that table 7 different concns nitrogen source fermentation obtains

During as nitrogen source fermentation, production of enzyme is higher take soy peptone, water-soluble plant protolysate, cottonseed meal and soybean cake powder.

Four, the optimization of Inorganic Salts and concentration

The formula of seed culture medium is with embodiment 2.

Basic medium forms (pH11) by solute and water, and described solute and the concentration in described seed culture medium thereof are as follows: glucose 10g/L, peptone 5g/L, yeast powder 5g/L and Na ₂CO ₃10g/L.

Fermention medium: add different inorganic salt or do not add inorganic salt on the basis of basic medium; Every kind of inorganic salt adopt different additions; Adopt respectively following inorganic salt: Trisodium Citrate, NaNO ₃, CaCl ₂, FeSO ₄7H ₂O, CuSO ₄5H ₂O, ZnCl ₂, KH ₂PO ₄, MgSO ₄

The results are shown in Table 8.

The production of enzyme that the fermentation of table 8 different concns inorganic salt obtains

Trisodium Citrate: can effectively promote strain enzyme-producing during 2-6g/L.NaNO ₃: strain enzyme-producing is not had obvious raising effect.CaCl ₂: can obviously improve the strain enzyme-producing amount.FeSO ₄7H ₂O: can promote strain enzyme-producing, promoter action is maximum when 1.2g/L.CuSO ₄5H ₂O: lower concentration can improve production of enzyme, and effect is optimum under the condition of 0.1g/L.ZnCl ₂: the ZnCl of trace ₂Just can suppress thalli growth, strain growth is obstructed and causes that enzymatic productivity descends.KH ₂PO ₄: can inorganic phosphorus be provided and play in substratum and regulate the pH effect for Growth of Cells, add 0.4-2.0g/L KH ₂PO ₄Be conducive to strain enzyme-producing.MgSO ₄: can promote strain enzyme-producing when 0.4-1.0g/L.

Embodiment 4, employing statistical analysis bacillus protein enzyme high-efficiency fermenting working condition

1, fractional factorial design (Fractional factorial design, FFD)

The optimum temperuture (34 ℃) of the strain fermentation of determining based on embodiment 3, optimal pH (pH11.0), the suitableeest kind age the (OD of seed liquor ₆₀₀Value is 12.0), the suitableeest inoculum size (2%), filter out optimum carbon source (Semen Maydis powder), optimum nitrogen source (soy peptone), can obviously promote the inorganic salt (MgSO of strain enzyme-producing ₄, KH ₂PO ₄And CaCl ₂), adopt 2 ^5-1The fractional factorial design screening affects the most important nutritive ingredient of strain enzyme-producing.In experiment, each nutritive ingredient is uneven as shown in table 9.

Table 9FFD experimental variable and level thereof

With Design Expert7.1.3 (Stat-Ease, Inc., Minneapolis, MN, USA) carry out experimental design and data analysis, design various nutritive ingredient combinations and corresponding enzyme activity output and see Table 10 (cultivation of bacterial strain is with the step 2 of embodiment 3).

Table 10FFD experimental design and result thereof

Result with Design Expert software his-and-hers watches 10 is carried out statistical study, obtain each factor and enzyme activity output and relational expression, see formula (I):

Y＝42930.56+7273.15x ₁-2041.67x ₂-358.33x ₃-547.22x ₄-84.26x ₅

+2682.41x ₁x ₂+791.67x ₁x ₃-178.7x ₁x ₄+217.59x ₁x ₅+425x ₂x ₃

-334.26x ₂x ₄+ 2.78x ₂x ₅+ 241.67x ₃x ₄-302.78x ₃x ₅+ 37.96x ₄x ₅Formula (I)

Wherein Y represents enzyme activity output, x ₁, x ₂, x ₃, x ₄And x ₅Represent respectively Semen Maydis powder, soy peptone, MgSO ₄, KH ₂PO ₄And CaCl ₂Encoded radio.Remove that in (1) formula, the p value greater than 0.05, does not have the polynomial expression of statistical significance, obtain representing the relational expression of factor and yield model, see formula (II):

Y=42930.56+7273.15x ₁-2041.67x ₂-547.22x ₄+ 2682.41x1x ₂+ 791.67x1x ₃Formula (II)

The F value of this model is 185.92, p＜0.0001, has statistical significance; R in addition ²=0.9986, adjusted R ²=0.9932 shows that equally First-Order Mode pattern (II) and FFD experiment have good dependency.

Through type (II) can find out, Semen Maydis powder is the most obvious to the influence of proteolytic enzyme output, soy peptone and KH ₂PO ₄Strain fermentation is produced enzyme also remarkably influenced, by steepest rising path, increases or reduce the concentration of these three main ingredients, can further improve enzyme activity output.

2, steepest rising experiment (Steepest ascent experiment)

The optimal conditions of FFD experiment operation according to a preliminary estimate usually away from the actual optimum point, the ability sufficient approximation real case in neighborhood that is right after that the response surface fit equation is only being investigated, have no similarity at other regional fit equation and the functional equation that is similar to, almost meaningless.So, could set up effective response surface fit equation after first approaching enzyme activity output maximum value zone.The gradient direction that the steepest hill climbing changes take experimental value is determined change step as the climbing direction according to the size of each factorial effect value in the first-order equation of FFD experimental fit, then tests along steepest rising path, until response value descends.Utilize the method, just approach rapidly the maximum response zone through 4 step experiments.Concrete experimental result table 11.

Table 11 steepest rising experimental result

The climbing gradient	The enzyme activity of 1mL crude enzyme liquid (U)
		1	50511.11±167.77
2	59377.78±167.77
		3	60577.78±828.21
4	60911.11±1108.22
		5	60177.78±1009.58
6	57711.11±504.79

3, central composite design (Central Composite Design) and response Surface Analysis

Response Surface Method is also referred to as Box-Wilson methodology, is a kind of comprehensive statistical analysis technique that is proposed by Box and Wilson.The method mainly realizes match and the assessment of response equation by central composite design (Central Composite Design, CCD).The impact that CCD obtains with the FFD experimental analysis is produced three principal elements of enzyme as variable, and each variable rises the vertex of experiment as mid point with steepest, and other secondary causes all remain on the mid point of FFD design.Design is comprised of 23=8 2 horizontal Factorial Design, 2 * 3=6 secondary axis test point or asterisk point and 6 central points.Wherein 23 Factorial Designs are used for the match first order modeling; Test point on coordinate axis or asterisk test point are used for the match second-order model and calculate factor interaction, and for the spinability of realizing designing, asterisk brachium value is 1.68; The test point, center is used for errot analysis.Factor and the level of experimental design are as shown in table 12.

Table 12CCD tests each level of factor and actual value

According to the factor in table 12 and level thereof, contain the CCD experiment (see Table 13 and Fig. 2) of 20 experiments with Design Expert7.1.3 design.According to the contrived experiment image data, each tests triplicate, and the mean value of getting three results carries out statistical study (cultivation of bacterial strain is with the step 2 of embodiment 3) as response value.

Table 13CCD experimental design and result thereof

Obtain being suitable for this central composite design second-order model by variance analysis (Analysis Of Variance, ANOVA) match, see formula (III):

Y = 61896.42 + 1071.66 x_{1} + 849.58 x_{2} + 552.13 x_{4} + 286.5 x_{1} x_{2} - 695 x_{1} x_{4}

- 514.25 x_{2} x_{4} - {728.66 x}_{1}^{2} - 1436.65 x_{2}^{2} - 375.28 x_{4}^{2}

Formula (III)

Wherein Y represents enzyme activity output, x ₁, x ₂And x ₄Represent respectively Semen Maydis powder, soy peptone, KH ₂PO ₄Concentration.This model F-Value=62.94, p＜0.0001; R ²=0.9827, Adj R ²=0.967.Ask the single order partial derivative to obtain x to formula (III) ₁, x ₂And x ₄Value be respectively 1.2,0.55 ,-0.76; Corresponding Semen Maydis powder, soy peptone, KH ₂PO ₄Concentration be respectively 18.9g/L, 6.1g/L, 1.1g/L, prediction enzyme activity output maximum can reach 62567U/mL.

The experimental verification of embodiment 5, bacillus protein enzymic fermentation model

Seed culture medium forms (pH11) by solute and water, and described solute and the concentration in described seed culture medium thereof are as follows: glucose 10g/L, peptone 5g/L, yeast powder 5g/L, KH ₂PO ₄1g/L, MgSO ₄0.2g/L and Na ₂CO ₃10g/L.

Fermention medium forms (pH11) by solute and water, and described solute and the concentration in described fermention medium thereof are as follows: Semen Maydis powder 18.9g/L, soy peptone 6.1g/L, Trisodium Citrate 4.0g/L, MgSO ₄0.4g/L, KH ₂PO ₄1.1g/L, CaCl ₂1.0g/L and Na ₂CO ₃10.0g/L.

1, bacterial strain BS036 is inoculated in seed culture medium, 34 ℃, 200rpm shaking culture are to the OD of bacterium liquid ₆₀₀Value reaches 12.0, obtains seed liquor.

2, the 1ml seed liquor is seeded in the 50ml fermention medium, 34 ℃, 200rpm shaking culture were collected fermented liquid every 3 hours, fermented 48 hours.

3, with the centrifugal 10min of fermented liquid 12000 * g, collect supernatant, be crude enzyme liquid.

4, detecting crude enzyme liquid lives as the enzyme of proteolytic enzyme and (adopts 60 ℃ as preset temperature: the 50mmol/L glycine-sodium hydrate buffer solution that adopts following damping fluid: pH10.0).

The enzyme work that each fermentation time obtains crude enzyme liquid sees Table 14.The polyacrylamide gel electrophoresis figure that each fermentation time obtains crude enzyme liquid sees Fig. 3.

Production of enzyme under the different fermentation time of table 14

Different fermentation times (hour)	The enzyme activity of 1mL crude enzyme liquid (U)
		3	97.50±36.86
6	482.50±82.61
		9	7740.00±789.05
12	19960.00±3704.52
		15	31125.00±4176.61
18	41737.50±3916.07
		21	47535.00±3860.45
24	55095.00±2515.41
		27	66170.00±3844.36
30	68950.00±3250.17
		33	66270.00±6275.74
36	64400.00±8249.70
		39	64840.00±687.41
42	46890.00±3745.13
		45	46310.00±6710.34
48	46430.00±5141.45

When utilizing the fermention medium fermentation 30h after optimizing, enzyme activity output is the highest, reaches 68950U/ml, near predictor 62567U/ml, has effectively confirmed the bacillus protein enzyme model.

Embodiment 6, utilize bacterial strain BS036 High-efficient Production proteolytic enzyme

One, utilize bacterial strain BS036 High-efficient Production proteolytic enzyme (batch 1)

Seed culture medium forms (pH11) by solute and water, and described solute and the concentration in described seed culture medium thereof are as follows: glucose 10.0g/L, peptone 5.0g/L, yeast powder 5.0g/L, KH ₂PO ₄1.0g/L, MgSO ₄0.2g/L and Na ₂CO ₃10g/L.

Fermention medium forms (pH11) by solute and water, and described solute and the concentration in described fermention medium thereof are as follows: Semen Maydis powder 18.0g/L, soy peptone 6.0g/L, Trisodium Citrate 2.0g/L, MgSO ₄0.1g/L, KH ₂PO ₄1.0g/L, CaCl ₂0.2g/L and Na ₂CO ₃10.0g/L.

(1) bacterial strain BS036 is inoculated in seed culture medium, 34 ℃, 200rpm shaking culture are to the OD of bacterium liquid ₆₀₀Value reaches 9.0, obtains seed liquor.

(2) the 1ml seed liquor is seeded in the 50ml fermention medium, 30 ℃, 200rpm shaking culture were collected fermented liquid every 3 hours, fermented 48 hours.

The results are shown in Table 15.

Production of enzyme under the table 15 different fermentations time (batch 1)

Different fermentation times (hour)	The enzyme activity of 1mL crude enzyme liquid (U)
		3	26.67±6.94
6	456.67±12.62
		9	6993.33±252.86
12	17440.00±551.40
		15	28300.00±482.63
18	37210.00±1517.90
		21	47650.00±1389.44
24	55870.00±856.52
		27	67266.67±728.02
30	77013.33±2622.89
		33	75173.33±2622.89
36	70480.00±2011.70
		39	70253.33±1800.76
42	50520.00±406.83
		45	46933.33±2482.30
48	48920.00±1558.18

Two, utilize bacterial strain BS036 High-efficient Production proteolytic enzyme (batch 2)

Seed culture medium forms (pH11) by solute and water, and described solute and the concentration in described seed culture medium thereof are as follows: glucose 10g/L, peptone 5g/L, yeast powder 5g/L, KH ₂PO ₄1g/L, MgSO ₄0.8g/L and Na ₂CO ₃10g/L.

Fermention medium forms (pH11) by solute and water, and described solute and the concentration in described fermention medium thereof are as follows: Semen Maydis powder 19.0g/L, soy peptone 6.5g/L, Trisodium Citrate 4.0g/L, MgSO ₄0.7g/L, KH ₂PO ₄1.1g/L, CaCl ₂1.0g/L and Na ₂CO ₃10.0g/L.

(1) bacterial strain BS036 is inoculated in seed culture medium, 30 ℃, 200rpm shaking culture are to the OD of bacterium liquid ₆₀₀Value reaches 10.0, obtains seed liquor.

(2) the 1ml seed liquor is seeded in the 50ml fermention medium, 34 ℃, 200rpm shaking culture were collected fermented liquid every 3 hours, fermented 48 hours.

The results are shown in Table 16.

Production of enzyme under the table 16 different fermentations time (batch 2)

Different fermentation times (hour)	Production of enzyme (U/mL)
		3	46.67±11.71
6	623.33±110.67
		9	8006.67±950.91
12	21573.33±1161.63
		15	32230.00±951.96
18	40730.00±2016.29
		21	45980.00±240.07
24	53130.00±564.00
		27	63586.67±1046.84
30	73853.33±2601.96
		33	75173.33±2622.89
36	70480.00±2011.70
		39	70253.33±1800.76
42	53040.00±392.82
		45	49146.67±685.25
48	51720.00±302.88

Three, utilize bacterial strain BS036 High-efficient Production proteolytic enzyme (batch 3)

Fermention medium forms (pH11) by solute and water, and described solute and the concentration in described fermention medium thereof are as follows: Semen Maydis powder 20.0g/L, soy peptone 7.0g/L, Trisodium Citrate 6.0g/L, MgSO ₄1.2g/L, KH ₂PO ₄1.2g/L, CaCl ₂1.2g/L and Na ₂CO ₃10.0g/L.

(1) bacterial strain BS036 is inoculated in seed culture medium, 34 ℃, 200rpm shaking culture are to the OD of bacterium liquid ₆₀₀Value reaches 12.0, obtains seed liquor.

The results are shown in Table 17.

Production of enzyme under the table 17 different fermentations time (batch 3)

Different fermentation times (hour)	Production of enzyme (U/mL)
		3	33.33±8.39
6	1336.67±71.83
		9	9913.33±495.55
12	24180.00±783.87
		15	32840.00±1007.59
18	46190.00±886.36
		21	48360.00±155.24
24	25340.00±1379.29
		27	63773.33±917.61
30	71053.33±1359.11
		33	65893.33±4072.99
36	68933.33±2349.29
		39	71200.00±3113.53
42	53426.67±1321.03
		45	54560.00±3097.47
48	52080.00±1678.15

Integrate, the conditional curve that 3 batches of production of enzyme that utilize bacterial strain BS036 High-efficient Production proteolytic enzyme change with fermentation time as shown in Figure 4.Genus bacillus BS036 extracellular protein production of enzyme when this fermentation condition bottom fermentation 27-40h can reach 60000-75000U/mL, and fermentation 30h reaches maximum enzyme output (75173U/mL).Utilize the method fermentative production cycle short, proteolytic enzyme output is high.In addition, genus bacillus BS036 still remains on the 50000U/mL left and right in fermentation late enzyme output, can stably manufactured proteolytic enzyme, keep the high activity and stability of proteolytic enzyme.Therefore, using fermentation condition that genus bacillus BS036 and optimization of the present invention obtains produces proteolytic enzyme and has not only that production of enzyme is high, fermentation period is short, low cost and other advantages, and removed it and be subject to katabolism regulation and control in the proteolytic enzyme fermentative production later stage, proteolytic enzyme production is very stable in whole fermenting process.

Claims

1. genus bacillus (Bacillus sp.) BS036, it is characterized in that: the deposit number of described bacterial strain is CGMCCNO.5178.

2. the method for the seed liquor of the described genus bacillus BS036 of preparation claim 1, comprise the steps: described genus bacillus BS036 is inoculated in following seed culture medium, and 26-37 ℃, 120-220rpm shaking culture obtain seed liquor;

Described seed culture medium is comprised of solute and water; Described solute and the concentration in described seed culture medium thereof are as follows: glucose 4-20g/L, peptone 3-8g/L, yeast powder 3-8g/L, KH ₂PO ₄0.5-1.5g/L, MgSO ₄0.1-1.0g/L and Na ₂CO ₃5-15g/L.

3. a method for preparing proteolytic enzyme, be the described genus bacillus BS036 of fermentation claim 1, obtains proteolytic enzyme.

4. method as claimed in claim 3, it is characterized in that: described method comprises the steps: the seed liquor that the described method of claim 2 prepares is inoculated in following fermention medium, and 26-37 ℃, 120-220rpm shaking culture obtain proteolytic enzyme;

Described fermention medium is comprised of solute and water; Described solute is comprised of carbon source, nitrogenous source and inorganic salt; Described carbon source is at least a in glucose, Semen Maydis powder, Zulkovsky starch, molasses, maltose and Testa Tritici; Described nitrogenous source is at least a in cottonseed meal, soybean cake powder, soy peptone, casein, water-soluble plant protolysate, fish peptone, yeast powder and Oxide peptone; Described inorganic salt are Trisodium Citrate, NaNO ₃, CaCl ₂, FeSO ₄, CuSO ₄, ZnCl ₂, KH ₂PO ₄, MgSO ₄And Na ₂CO ₃In at least a.

5. the application of the described genus bacillus BS036 of claim 1 in producing proteolytic enzyme.