CN104357503A - Method for improving yield of isoleucine - Google Patents

Abstract

The invention discloses a method for improving yield of isoleucine. A methionine and lysine dual defect isoleucine producing bacterial strain B.flavum KM1 is bred through ultraviolet mutation breeding; and the original strain of the bacterial strain is B.flavumi 10505(Met<->+Eth<r>+alpha-AB<r>+AEC<r>). VB12 is added to a seed culture medium and a fermentation medium of the isoleucine producing bacteria B.flavum KM1, so that the seed vigor is improved, and the fermentation cycle is shortened, thus the yield of L-isoleucine is increased.

Description

A kind of method improving Isoleucine output

Technical field

The invention belongs to biological technical field, relate to the fermentation processes of ILE producing strains, be applied to Studies on Fermentation of L-isoleucine.

Background technology

Isoleucine is also known as " L-iLeu ", and systematic naming method is " α-amino-γ-methylthio-n-butyric acid ", hydrophobicity branched-chain amino acid.Because having two unsymmetrical carbons in α position and β position, thus have that D, L, D are other, the other four kinds of optically active isomers of L, what occurring in nature existed is only ILE, and its excess-three kind is all without nutritive value.ILE is one of human body 8 kinds of indispensable amino acids, has the status of particularly important in human life's metabolism.There is the function promoting the synthesis of body internal protein, enzyme and peptide hormone, very important in Protein metabolism, be mainly used in treating neurological disorder, appetite stimulator and anaemia, have the function strengthening body immunity.Owing to there are four kinds of optically active isomers, be therefore difficult to the method adopting chemical synthesis or chemosynthesis to combine with enzyme process, the ILE that cheap manufacture purity is high, only have employing fermentation method.

The key of ILE suitability for industrialized production is fermentation, and the height of fermentation level is the principal element determining product cost.Improve the output of ILE, not only want a strain to be applicable to the excellent superior strain of suitability for industrialized production, and need and best technological condition for fermentation that this bacterial strain matches and control device.

Summary of the invention

The object of the invention is to solve in ILE suitability for industrialized production, low, the problem that production cost is high of fermentation level, and provide a kind of method improving Isoleucine output, the present invention selects a strain Isoleucine Producing Mutant of Brevibacterium flavum by UV mutation b.flavumkM1, by adding VB ₁₂to ILE producing strains brevibacterium flavum brevibacterium flavumkM1(Met ^-+ Lys ^-+ Eth ^r+ α-AB ^r+ AEC ^r) seed culture condition and fermenting process be optimized, thus further improve the output of ILE.

VB ₁₂i.e. cobalami, containing cobalt and corrin in molecule, corrin the 6th coordination in conjunction with other groups, can produce various cobalami, comprises the hydrogen cobalami be combined with hydrogen, the methyl cobalamin be combined with methyl, the coenzyme B that is combined with 5 '-Desoxyadenosine ₁₂deng.Methyl cobalamin can be used as methyl carrier, accepts the methyl that methyl tetrahydrofolate provides, for the synthesis of methionine(Met).Some rely on coenzyme B ₁₂enzyme catalysis 1,2 move molecular rearrangement reaction, i.e. the metathesis reaction of hydrogen atom and a certain group on adjacent carbons.Such as in propionic acid metabolism, catalysis methylmalonyl CoA changes the mutase of succinyl-coenzyme A into just with coenzyme B ₁₂for cofactor.In addition, VB ₁₂also participate in the synthesis of picodna (DNA), the metabolism of carbohydrate and protein, increase the synthesis of nucleic acid and protein.

The method of the present invention is:

A strain ILE producing strains is selected by UV mutation b.flavumkM1, the starting strain of this bacterial strain is brevibacterium flavum b. flavumi10505 (Met ^-+ Eth ^r+ α-AB ^r+ AEC ^r).

By adding VB ₁₂the seed culture condition of KM1 bacterial strain and fermenting process are optimized.

Described brevibacterium flavum b.flavumi10505 bacterium source is biological and Agricultural engineering institute fermentation engineering laboratory in Jilin University.

Described ILE producing strains b.flavumkM1, is applied to Studies on Fermentation of L-isoleucine.

Accompanying drawing explanation

Fig. 1 is the product acid preferably ILE producing strains that mutagenic and breeding goes out, and wherein i10505 is starting strain;

Fig. 2 is the growth curve of KM1 bacterial strain, adds VB ₁₂vB is added with nothing ₁₂contrast;

Fig. 3 is VB in seed culture medium ₁₂addition pair b.flavumkM1 bacterial strain produces the influence curve figure of acid.

Fig. 4 is VB in fermention medium ₁₂addition pair b.flavumkM1 bacterial strain produces the influence curve figure of acid.

Embodiment

The present invention selects a strain methionine(Met) and Methionin double defect type Isoleucine Producing Mutant of Brevibacterium flavum bacterial strain by UV mutation b.flavumkM1, the starting strain of this bacterial strain is b. flavumi10505 (Met ^-+ Eth ^r+ α-AB ^r+ AEC ^r).

By at Isoleucine Producing Mutant of Brevibacterium flavum b.flavumvB is added in the seed culture medium of KM1 He in fermention medium ₁₂, improve seed vitality, shorten fermentation period, thus improve ILE output.

The present invention is with brevibacterium flavum b.flavumi10505 is starting strain, brevibacterium flavum b.flavumi10505 bacterium source is biological and Agricultural engineering institute fermentation engineering laboratory in Jilin University.

Concrete steps of the present invention are as follows:

(1) UV mutation:

A. ultraviolet mutagenesis

With brevibacterium flavum b.flavumi10505 is starting strain, the Spawn preparation bacteria suspension of phase of taking the logarithm, and carries out microscopic counting control cell concentration and is about 1 × 10 ⁸individual/mL.Get the bacteria suspension prepared, adopt 15W ultraviolet lamp, irradiation distance 30cm, measures the sterilizing rate of different irradiation time, draws lethality rate curve.Suitable mutagenic condition is selected to carry out ultraviolet mutagenesis.

B. the screening of auxotrophic mutation bacterial strain

Middle cultivation: get the bacterium liquid 100 μ L after mutagenic treatment and be coated with CM flat board, cultivate 48 h for 31 DEG C.CM substratum (g/L): yeast powder 5, peptone 10, extractum carnis 10, corn steep liquor 20mL, agar 15.

Eliminate wild-type: with sterilizing toothpick picking list bacterium colony, one_to_one corresponding is inoculated in CM flat board and MM flat board, cultivates 48 h for 31 DEG C.Select only at the bacterial strain of CM grow on plates.MM substratum (g/L): glucose 20, ammonium sulfate 10, KH ₂pO ₄3H ₂o 1, MgSO ₄7H ₂o 0.4, FeSO ₄7H ₂o 0.01, MnSO ₄h ₂o 0.01, VH 0.1mg, VB ₁0.1mg, agar 20.

Auxotroph is identified: be connected in the supplemental medium containing Met+Lys+Ala, Met+Lys, Met+Ala, Lys+Ala by the bacterial strain selected correspondence, cultivates 48 h, determines defective type for 31 DEG C.

Shaking flask primary dcreening operation and multiple sieve: select two to lack or three bacterial strains lacked carry out preliminary product acidity test, therefrom select the bacterial strain producing ILE, further study.The bacterial strain selected is carried out Shake flask batch fermentation, therefrom selects the bacterial strain that product acid activity is higher.Produce sour result as shown in Figure 1, in Fig. 1, the defective type of each bacterial strain is: i10505(Met-); AM1(Ala-+Met-); AM2(Ala-+Met-); KM1(Met-+Lys-); KM2(Met-+Lys-).Seed culture medium (g/L): glucose 35, ammonium sulfate 3, KH ₂pO ₄3H ₂o 1.5, MgSO ₄7H ₂o 0.4, FeSO ₄7H ₂o 0.01, MnSO ₄h ₂o 0.01, corn steep liquor 15mL, soya-bean cake hydrolyzed solution 15 mL, VH 0.5mg, VB ₁2.5mg, urea 2(divide disappear).Fermention medium (g/L): glucose 80, ammonium sulfate 15, KH ₂pO ₄3H ₂o 1.5, K ₂hPO ₄3H ₂o 3, MgSO ₄7H ₂o 0.4, FeSO ₄7H ₂o 0.015, MnSO ₄h ₂o 0.015, corn steep liquor 25mL, soya-bean cake hydrolyzed solution 20mL, VH 0.5mg, VB ₁2.5mg, Ala 20mg, Met 20mg, Lys 20mg, CaCO ₃20(divide disappear).31 DEG C, seed, 200 r/min cultivate 24h.10% inoculum size sending and receiving ferment, 31 DEG C, 200 r/min fermentation culture 48h.

Finally select a strain and produce the bacterial strain KM1 that acid is higher, heritability is stable, further fermentation optimization is carried out to it.

(2) conditions of flask fermentation optimization:

C. seed culture optimization

Activated inclined plane is cultivated b.flavumkM1, activation medium (g/L): peptone 1, yeast leaching powder 0.5, extractum carnis 1, corn steep liquor 2mL, agar 1.5.

Draw from activated inclined plane and get a ring bacterium, be connected in seed culture medium, 31 DEG C, 200 r/min cultivate.First, utilize uniform design to be optimized seed culture medium, the seed culture based component after optimization is (g/L): glucose 30, ammonium sulfate 3, trimethyl-glycine 1.5, KH ₂pO ₄3H ₂o 1.5, MgSO ₄7H ₂o 0.4, FeSO ₄7H ₂o 0.01, MnSO ₄h ₂o 0.01, corn steep liquor 15mL, soya-bean cake hydrolyzed solution 20 mL, VH 0.5mg, VB ₁2.5mg.Then, be optimized seed culture condition, obtaining optimal culture conditions is 31 DEG C, 200 r/min, 24h.

On this basis, by adding VB in seed culture medium ₁₂, optimize seed culture condition further.As shown in Figure 2, VB is added ₁₂the growth time that rear seed reaches logarithmic phase shortens, and about 12h, control group (does not add VB ₁₂) be 24h.After sending and receiving ferment, the output of ILE also increases.As shown in Figure 3, the output of control group (addition is " 0 ") ILE is 8.44 g/L.VB in seed culture medium ₁₂when addition is 8 mg/L, ILE output is up to 9.66 g/L, improves 14.4% than control group.

D. Medium of shaking flask fermentation optimization

b.flavumkM1 bacterial strain is amino acid-deficient, first carries out orthogonal optimization to the addition of L-Ala, Methionin, methionine(Met) in fermention medium.On this basis, by adding VB in the fermentation medium ₁₂, shorten fermentation period, raise the efficiency.80g sugar batch fermentation is about 35h, is 48h before optimizing.By VB in research substratum ₁₂addition improve the output of ILE.As shown in Figure 4, the output of control group ILE is 9.99 g/L.VB in fermention medium ₁₂when addition is 2mg/L, ILE output is up to 11.83g/L, improves 18.4% than control group.

E.5L fermentor tank fed-batch fermentation

By fermentation cylinder for fermentation substratum through about 121 DEG C, 15min steam sterilizing, access bacterial classification after being cooled to about 31 DEG C, inoculum size is 8%; Simultaneously by through overdraft, filter after sterile air input fermentor tank in, by through 121 DEG C, 800g/L Glucose Liquid after 8min steam sterilizing carries out Continuous Flow and adds; PH7, dissolved oxygen 10 ~ 20%, fermentation 50h; Produce acid and reach 38.9g/L.With do not add VB ₁₂compare, fermentation time reduction 10h, produces acid and improves 14.8%.

F. ILE content in Fermentation Liquor by High Performance Liquid Chromatography is utilized.

Claims (3)

1. improve a method for Isoleucine output, the method is: select a strain Isoleucine Producing Mutant of Brevibacterium flavum by UV mutation b.flavumkM1, the starting strain of this bacterial strain is brevibacterium flavum b. flavumi10505(Met ^-+ Eth ^r+ α-AB ^r+ AEC ^r); At Isoleucine Producing Mutant of Brevibacterium flavum b.flavumvB is added in the seed culture medium of KM1 He in fermention medium ₁₂.

2. a kind of described method improving Isoleucine output according to claim 1, is characterized in that: described brevibacterium flavum b.flavumi10505 bacterium source is biological and Agricultural engineering institute fermentation engineering laboratory in Jilin University.

3. a kind of described method improving Isoleucine output according to claim 1, is characterized in that: described Isoleucine Producing Mutant of Brevibacterium flavum b.flavumkM1 is applied to Studies on Fermentation of L-isoleucine.