CN109825466A - A kind of method that straight chain maltopentaose generates enzyme secreting, expressing in bacillus subtilis - Google Patents

Abstract

The invention discloses a kind of methods that straight chain maltopentaose generates enzyme secreting, expressing in bacillus subtilis, belong to technical field of enzyme engineering.The present invention optimizes the conditions of flask fermentation of genetic engineering bacterium by constructing the bacillus subtilis secretion expression system of G5- amylase, finally determines preferable culture medium composition are as follows: yeast powder 30g/L, cornstarch 6g/L, KH₂PO₄2.3g/L, K₂HPO₄12.5g/L, pH 7.5.At 37 DEG C of fermentation temperature, enzyme activity reaches 451.1U/mL after shaken cultivation 48h under 200r/min, improves 127.5% than the enzyme activity level under initial culture conditions, improves 10.9 times than document report level.

Description

A kind of method that straight chain maltopentaose generates enzyme secreting, expressing in bacillus subtilis

Technical field

The present invention relates to a kind of methods that straight chain maltopentaose generates enzyme secreting, expressing in bacillus subtilis, belong to enzyme Field of engineering technology.

Background technique

Maltopentaose (G5) is small molecule made of passing through α-Isosorbide-5-Nitrae-glucosides key connection as unit using α-D- glucopyranose, It is one kind of linear maltooligosacchaeides.G5 has good food processing adaptability: can be used as the soft sweetener of mouthfeel；It is drying It can be used as moisture adjuster in roasting, dilated food；The thawing resistance energy of cold drink can be improved in cold drink, improve the expansion of ice cream Rate；Crystal structure can be effectively suppressed in the food such as chocolate, jam；It can be used as thickener in supping；Furthermore can also Inhibit the protein denaturation in age of starch and quick-frozen food, to extend the shelf life.

G5 also has following unique physiological function: it is digested absorption in small intestine, and caused blood glucose response compares glucose Steadily, it slowly can constantly be energized for human body, can be used for energy supplement or the pancreatectomy patient, nephrotic of sportsman Dietary therapy；It is not easy to be utilized by bacterial fermentation, is conducive to pre- anti-caries；It can selective depression enteron aisle spoilage organisms growth and Promote proliferation of probiotics, to safeguard intestinal health；Absorption of the human body to calcium, effectively the prevention middle-aged and the old can additionally be promoted Osteoporosis.Therefore, G5 has good application prospect in arsenic, infant food and health food.

The industrialized production of G5 and the production method of other linear maltooligosacchaeides are similar, are to pass through linear maltooligosacchaeides Generation enzyme hydrolysis starch α -1,4- glycosidic bond cuts multiple glucose units and forms.The side of existing production linear maltooligosacchaeides Method is monopolized by Japan, it usually needs and alpha-amylase first carries out starch liquefacation and is saccharified again by the oligomeric generation enzyme of straight chain malt, and Calcium ion is added to improve the stability of enzyme, and the country not yet realizes the industrialized production of linear maltooligosacchaeides.

Current domestic and international production linear maltooligosacchaeides research report in, concentration of substrate used it is lower (1~ 5%), transform level is generally 50%~75% or so；In the existing research for G5- amylase, the G5 in product contains Amount only has qualitative analysis and does not do quantitative explanation, not directly expand and be applied to industrialized production.As straight chain malt is oligomeric Application prospect of the sugar in every field is more and more extensive, needs to develop the high-purity with industrial application value at home single Linear maltooligosacchaeides.

Key enzyme of the G5- amylase as production G5, develops G5- amylase efficient secretory expression in bacillus subtilis Method the production level of China G5 can be improved, reduce application cost, and the production for other linear maltooligosacchaeides With guidance and reference, and the comprehensive high-efficiency of cornstarch is utilized and is of great significance.

Summary of the invention

Technical problem to be solved by the present invention lies in G5- amylase Escherichia coli (Escherichia coli, E.coli the expression efficiency in) is low, and E.coli expression system is not suitable for producing food-grade G5- starch there are pathogenic Enzyme.For a kind of method that this problem proposes G5- amylase secreting, expressing in bacillus subtilis, by deriving from The G5- amylase coding base for producing G5 of Bacillus stearothermophilus (B.stearothermophilus) Because carrying out clonal expression in bacillus subtilis, to establish a kind of G5- amylase efficient secretion table in bacillus subtilis The method reached.

The first purpose of the invention is to provide a kind of recombined bacillus subtilis, be in bacillus subtilis for place Main, expression straight chain maltopentaose generates enzyme；The straight chain maltopentaose generates amino acid sequence shown in enzyme ID containing SEQ NO.1 Column.

In one embodiment of the invention, coding straight chain maltopentaose generates the nucleotide sequence of the gene of enzyme such as Shown in SEQ ID NO.2.

In one embodiment of the invention, using bacillus subtilis WB600 as expressive host.

In one embodiment of the invention, using pST as expression vector.

A second object of the present invention is to provide a kind of method for constructing above-mentioned recombined bacillus subtilis, feature exists In, which comprises

(1) using Bacillus stearothermophilus genomic DNA as template, both ends point are obtained by PCR amplification G5 target gene fragment not containing I restriction enzyme site of Nco I and Xho；

(2) by the g5 genetic fragment insertion pMD 18-T simple plasmid obtained in (1), cloning vector pMD is obtained 18-T simple/g51；

(3) it using I site Nco inside target gene in rite-directed mutagenesis removal pMD 18-T simple/g51, is carried Body pMD 18-T simple/g52；

(4) carrier pMD 18-T simple/g52 recycles the purpose containing cohesive end after I double digestion of Nco I and Xho G5 genetic fragment is simultaneously inserted into pST plasmid, and pST/g5 plasmid is obtained, and expression vector pST/g5 is converted host bacillus subtilis WB600 obtains genetic engineering bacterium.

Third object of the present invention is to provide a kind of methods for producing straight chain maltopentaose and generating enzyme, using above-mentioned recombination Bacillus subtilis is fermented.

In one embodiment of the invention, fermentation medium used are as follows: yeast powder 30-36g/L, cornstarch 5- 7g/L, KH₂PO₄2.0-2.5g/L, K₂HPO₄12-13g/L, pH are 7.0~8.0.

In one embodiment of the invention, fermentation medium used are as follows: yeast powder 30g/L, cornstarch 6g/L, KH₂PO₄2.3g/L, K₂HPO₄12.5g/L, pH 7.5.

In one embodiment of the invention, it is needed before fermentation by seed activation, seed activation includes: will be above-mentioned heavy Group bacillus subtilis is inoculated into LB culture medium, and in 35-38 DEG C, 200-220r/min cultivates 10-12h.

In one embodiment of the invention, the seed liquor after activation is forwarded to fermentation with the inoculum concentration of 1%-5% In culture medium, in 35-38 DEG C, 200-220r/min cultivates 48-96h.

Fourth object of the present invention is to provide above-mentioned recombined bacillus subtilis and is preparing maltopentaose or containing malt five Application in the product of sugar.

Beneficial effects of the present invention:

The present invention shakes genetic engineering bacterium by constructing the bacillus subtilis secretion expression system of G5- amylase Bottle fermentation condition optimizes, and finally determines preferable culture medium composition are as follows: yeast powder 30g/L, cornstarch 6g/L, KH₂PO₄ 2.3g/L, K₂HPO₄12.5g/L, pH 7.5.At 37 DEG C of fermentation temperature, enzyme activity reaches after shaken cultivation 48h under 200r/min 451.1U/mL improves 127.5% than the enzyme activity level under initial culture conditions, improves 10.9 times than document report level. The present invention realizes the straight chain maltopentaose generation enzyme from B.stearothermophilus in Bacillus subtilis Efficient secretory expression in WB600, i.e. B.subtilis WB600 is conducive to break external monopolization, improves China G5 and produces water It is flat, application cost is reduced, and there is guidance and reference for the production of other linear maltooligosacchaeides, and for corn The comprehensive high-efficiency of starch is utilized and is of great significance.

Detailed description of the invention

Fig. 1 is the building process of expression vector pST/g5.

Fig. 2 is the growth curve of LB culture media shaking vase fermentation B.subtilis WB600 (pST/g5).

Fig. 3 is influence of the type of culture medium to G5- amylase shake flask fermentation of setting out of fermenting.Wherein, 1:LB；2:TSB；3: TB；4: preliminary fermentation culture medium；5:M9.Each value is the average value of 3 independent experiments, and different letters indicate exist on histogram Significant difference (P < 0.05).

Fig. 4 is the influence of fermentation temperature and time to G5- amylase shake flask fermentation.

Fig. 5 is influence of the initial pH of fermentation to G5- amylase shake flask fermentation.

Fig. 6 is influence of the different carbon source to engineering bacteria fermentation producing enzyme.Wherein, 0: being not added with；1: glycerol；2: potato Starch；3: cornstarch；4: tapioca；5: maltodextrin；6: glucose；7: maltose；8: sucrose.Each value is only three times The average value of experiment is found, different letters indicate that there are significant difference (P < 0.05) on histogram.

Fig. 7 is influence of the cornstarch concentration to G5- amylase enzyme shake flask fermentation.Wherein, each value is independent real three times The average value tested, different letters indicate that there are significant difference (P < 0.05) on histogram.

Fig. 8 is the inorganic nitrogen-sourced influence to G5- amylase shake flask fermentation.Each value is the average value of independent experiment three times, Different letters indicate that there are significant difference (P < 0.05) on histogram.

Fig. 9 is influence of the organic nitrogen source to G5- amylase shake flask fermentation.Wherein, 1: being not added with；2: tryptone；3: big Legumin peptone；4: yeast powder；5: yeast extract；6: fish meal protein peptone；7: beef extract.Each value is averaged for independent experiment three times It is worth, different letters indicate that there are significant difference (P < 0.05) on histogram.

Figure 10 is influence of the dusty yeast concentration to G5- amylase shake flask fermentation.Wherein, each value is independent experiment three times Average value, different letters indicate that there are significant difference (P < 0.05) on histogram.

Specific embodiment

(1) extraction, verifying and recycling of DNA

The extracting method of B.stearothermophilus genomic DNA, using agarose gel electrophoresis to the matter of extraction Referring to Southern Yangtze University Lee Zhao Feng Ph.D. Dissertation, (Li Zhaofeng softens series bacillus α-ring and pastes the method that grain DNA is verified Expression and its product specificities analysis [D] Southern Yangtze University of the smart glucosyltransferase in Escherichia coli, 2009).

Plasmid is extracted using the small amount plasmid extraction kit (article No.: B518191) of Shanghai Sheng Gong bio-engineering corporation DNA, specific extracting method is referring to specification.

Using the plastic recovery kit (article No.: D0056) of the green skies biotechnology research institute in Shanghai from Ago-Gel Target gene fragment is recycled, concrete operation method is referring to specification.

(2) preparation and conversion of competent cell

It is prepared using the super competence reagent preparation box (article No.: B529303) of Shanghai Sheng Gong bio-engineering corporation E.coli competent cell, method are learned referring to specification, the method for transformation of competent cell referring to Southern Yangtze University doctor Li Zhaofeng Degree thesis whole-length.

The preparation of B.subtilis competent cell and method for transformation (are opened referring to Southern Yangtze University's Zhang Jiayu master thesis Expression [D] .2010 of good fine jade bacillus macerans alpha-cyclodextrin glucosyl transferase in Pichia pastoris and hay bacillus).

(3) clone of G5- amylase gene

Using B.stearothermophilus genomic DNA as template, is separately designed and contained according to the sequence of G5- amylase The both ends primer (Jin Weizhi Biotechnology Co., Ltd synthesizes by Suzhou) of I restriction enzyme site of Nco I and Xho, through Prime G5 gene is obtained after the amplification of STAR HS archaeal dna polymerase.

PCR operating method is referring to Southern Yangtze University Lee Zhao Feng Ph.D. Dissertation.

Positive and negative design of primers is as follows:

It is positive: 5 '-CATGCCATGGCCGCACCGTTTAAC-3 ' (underscore indicates I restriction enzyme site of Nco)

It is reversed: 5 '-CCGCTCGAGAGGCCATGCCACCAA-3 ' (underscore indicates I restriction enzyme site of Xho)

By g5 gene cloning into pMD 18-T simple plasmid after obtain cloning vector, Transformed E .coli JM109, sieve Choosing is further cultivated after obtaining positive strain, extracts plasmid pMD 18-T simple/g51, and carry out restriction enzyme digestion and electrophoresis and sequencing is tested Card is completed wherein being sequenced by Suzhou Jin Weizhi Biotechnology Co., Ltd.

(4) mutation in I site Nco

Using plasmid pMD 18-T simple/g51 as template, design complementary primer is (limited by Suzhou Jin Weizhi biotechnology Company's synthesis), one-step mutation is carried out to I site Nco in g5 gene under the premise of guaranteeing that amino acid classes are constant.

PCR operating method is referring to Southern Yangtze University Lee Zhao Feng Ph.D. Dissertation.

Design of primers is as follows:

It is positive: 5 '-ATGGGTCGACCCGTGGTTCAAACC-3 ' (underscore base indicates mutating alkali yl)

It is reversed: 5 '-GGTTTGAACCACGGGTCGACCCAT-3 ' (underscore base indicates mutating alkali yl)

Variants Transformed E .coli JM109 after the processing of Dpn I is screened and is extracted plasmid pMD after cultivating positive strain 18-T simple/g52 is simultaneously verified.

(5) building and conversion of expression vector pST/g5

Plasmid pMD 18-T simple/g52 recycles the purpose base containing cohesive end after I double digestion of Nco I and Xho Because of segment, and with T4DNA ligase by itself and the pST plasmid crossed through identical restriction endonuclease and alkaline phosphatase treatment Segment connects, and is applied on the LB agar plate containing 5 μ g/mL kanamycins after Transformed E .coli JM109, cultivates in 37 DEG C 10~12h, picking single colonie cultivate 10~12h in the LB culture medium containing identical resistance.Plasmid and through sequence verification is extracted, is obtained The expression vector pST/g5 of correct g5 gene order must be contained, then convert host bacillus subtilis B.subtilis WB600, obtained Obtain the genetic engineering bacterium B.subtilis WB600 (pST/g5) that can express g5 gene.

(6) seed activation

The kanamycins of final concentration of 5 μ g/mL is added in the 250mL conical flask equipped with 50mL LB culture medium, then moves The glycerol tube bacterial strain B.subtilis WB600 (pST/g5) for taking 100 μ L to be preserved in -80 DEG C, is connected in bottle, in 37 DEG C of shaking tables It cultivates 10~12h (revolving speed 200r/min).

(7) fermented and cultured

The kanamycins of final concentration of 5 μ g/mL is added in the 250mL conical flask equipped with 50mL fermentation medium, then Seed liquor (OD after pipetting 2mL activation₆₀₀It is 1.2 or so) it is forwarded in bottle, 48-96h is cultivated in 37 DEG C of shaking tables, and (revolving speed is 200r/min).After stopping fermentation, it is centrifuged fermentation liquid 15min under the conditions of 4 DEG C, 10000 × g, collects supernatant and obtains G5- The crude enzyme liquid of amylase.

(8) definition and detection method of enzyme activity

The definition of enzyme activity: 1 enzyme activity is defined as to generate enzyme amount needed for 1 μm of ol reduced sugar (with glucose meter) per minute Unit (U).

The detection method of enzyme activity: the measurement of enzyme activity is recombinated using 3,5- dinitrosalicylic acid (DNS) method.With C₆H₈O₇- Na₂HPO₄Buffer (10mM, pH 5.5) prepares 1% (w/v) soluble starch solution as substrate, adds in 1.98mL substrate Enter the enzyme solution after 20 μ L dilute, react 15min at 60 DEG C, 2.0mL DNS solution is added and terminates reaction, develop the color 5min in boiling water bath Ice bath is cooling immediately afterwards, measures light absorption value under 540nm, calculates content of reducing sugar in system according to glucose standard curve.

Embodiment 1

The building process of expression vector pST/g5 is as shown in Figure 1.Using B.stearothermophilus genomic DNA as mould Plate obtains purpose g5 genetic fragment of the both ends respectively containing I restriction enzyme site of Nco I and Xho by PCR amplification, and is inserted Enter in pMD 18-T simple plasmid, cloning vector pMD 18-T simple/g51 is obtained, to improve subclone effect.Through Analysis finds, there is also identical I sites Nco at about 1029bp inside the target gene, in order to more accurately carry out Double digestion removes restriction enzyme site at this using rite-directed mutagenesis, obtains carrier pMD 18-T simple/g52, and through digestion and survey Sequence verifying.

Carrier pMD 18-T simple/g52 recycles the purpose g5 containing cohesive end after I double digestion of Nco I and Xho Genetic fragment is simultaneously inserted into pST plasmid, and pST/g5 plasmid is obtained, and the secreted expression carrier pST/g5 through sequence verification is converted Host B.subtilis WB600 is obtained genetic engineering bacterium B.subtilis WB600 (pST/g5).

Embodiment 2

Appropriate genetic engineering bacterium B.subtilis WB600 is accessed in the 250mL shaking flask equipped with 50mL LB culture medium (pST/g5), different time sampling is cultivated under the conditions of 37 DEG C, 200r/min, measures cell concentration, as a result as shown in Figure 2.It can See that 0~6h is lag phase, 6~14h is logarithmic growth phase, and the stage of stable development is entered after 14h.The thallus adaptability of logarithmic growth phase and Fertility is stronger, possesses higher activity, can be used as seed liquor for enzymatic production.

Embodiment 3

By B.subtilis WB600 (pST/g5) seed liquor activated through LB culture medium be respectively connected to five kinds it is more typical It is cultivated for 24 hours, as a result as shown in Figure 3 in B.subtilis culture medium (LB, TSB, M9, TB, preliminary fermentation culture medium).From enzyme activity water Preferable fermentation medium is TB culture medium from the point of view of putting down, and enzyme activity for 24 hours of fermenting at 37 DEG C is significantly better than other trainings up to 198.3U/mL Support base；And with M9 culture medium culturing gene engineering bacteria, then effect is poor, and enzyme activity is only 16.9U/mL under the same terms.

Embodiment 4

Seed liquor after activation is forwarded in TB culture medium, respectively at shaken cultivation in 25 DEG C, 30 DEG C, 37 DEG C of shaking tables, Enzyme activity is measured by fermentation time spot sampling, as a result as shown in Figure 4.As seen from the figure, the enzyme activity of 37 DEG C of condition fermentation crude enzyme liquids The enzyme activity being apparently higher than under the conditions of 30 DEG C and 25 DEG C, ferment 48h after enzyme activity reach 338.0U/mL, be the lower 30 DEG C of shaking tables of same time Middle fermentation 2.6 times of enzyme activity, 4.5 times of enzyme activity of fermenting in 25 DEG C of shaking tables；Ferment 48~96h, and enzyme activity growth rate slows down, gradually Stablize.

Embodiment 5

During the fermentation, the vital movement of bacterium is influenced by pH, and its metabolite can lead to medium pH Variation, therefore the pH during shake flask fermentation not easy to control, can only optimize the pH of culture medium when fermenting initial.By the kind after activation Sub- liquid is respectively connected in the TB culture medium of different pH, measures enzyme activity after the 48h that ferments at 37 DEG C, as a result as shown in Figure 5.As a result table Bright genetic engineering bacterium enzymatic production level under conditions of neutral meta-alkali (pH 7.0~8.0) is higher, and preferably ferment initial pH It is 7.5, corresponding enzyme activity is up to 353.6U/mL.When the initial pH that ferments is larger in acid or alkalinity, then enzyme activity significantly reduces.

Embodiment 6

On the basis of TB culture medium, referring to the phosphorus content of glycerol, respectively with glycerol, potato starch, cornstarch, wood Sweet potato starch, maltodextrin (DE 4), glucose, maltose, sucrose probe into it to engineering bacteria fermentation producing enzyme water as carbon source Flat influence, as a result as shown in Figure 6.The result shows that using potato starch, cornstarch, tapioca and maltodextrin as carbon When source, enzymatic production level compares using glycerol as carbon source Shi Genggao, wherein enzyme activity is reachable when using potato starch as carbon source 392.8U/mL is that carbon source improves 23.6% than glycerol, is then distinguished using cornstarch, tapioca and maltodextrin as carbon source Improve 16.6%, 19.6% and 10.6%.Enzyme activity when due to three kinds of starch being carbon source is horizontal, and there was no significant difference, practical raw The cornstarch that advantage of lower cost may be selected in production is carbon source.When using sucrose and maltose as carbon source, enzyme activity be not so good as with Enzyme activity when glycerol is carbon source is horizontal.And glucose then obviously inhibits the enzymatic production of genetic engineering bacterium horizontal.

Embodiment 7

It selects the cornstarch of advantage of lower cost as carbon source, further carbon source concentration is optimized, as a result such as Fig. 7 It is shown.As it can be seen that with cornstarch be G5- amylase shake flask fermentation carbon source when, additive amount be the corresponding enzyme activity of 6g/L it is higher (402.4U/mL)。

Embodiment 8

It is fermented with containing inorganic nitrogen-sourced culture medium to genetic engineering bacterium, as seen from Figure 8, is measured in fermented supernatant fluid Enzyme activity it is very low, when using 8g/L urea as nitrogen source, enzyme activity is relatively high, but also only up to 40.7U/mL.And have as seen from Figure 9 Machine nitrogen source is obviously particularly suited for the enzymatic production of genetic engineering bacterium (additive amount is 24g/L).Genetic engineering bacterium is to tryptose The utilizing status of peptone is relatively poor, enzyme activity 210.6U/mL；And yeast powder is preferable nitrogen source, genetic engineering bacterium enzymatic activities can Up to 405.8U/mL, differed not with the producing enzyme of TB culture medium under the same terms (using cornstarch as carbon source) horizontal (407.6U/mL) Greatly, this also in side light TB culture medium tryptone the enzymatic production of genetic engineering bacterium is acted on it is little.

Embodiment 9

Nitrogen concentration is also factor in need of consideration of fermenting.As seen from Figure 10, when using yeast powder as nitrogen source, preferable nitrogen source Concentration is 30g/L, and corresponding enzyme activity is up to 451.1U/mL, when further increasing nitrogen concentration to 36g/L, genetic engineering bacterium Enzymatic production level and when 30g/L without significant difference.

Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not intended to limit the invention, any to be familiar with this skill The people of art can do various change and modification, therefore protection model of the invention without departing from the spirit and scope of the present invention Enclosing subject to the definition of the claims.

SEQUENCE LISTING

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Claims (10)

1. a kind of recombined bacillus subtilis, which is characterized in that be, for host, to express straight chain malt five in bacillus subtilis Sugar generates enzyme；The straight chain maltopentaose generates amino acid sequence shown in enzyme ID containing SEQ NO.1.

2. recombined bacillus subtilis according to claim 1, which is characterized in that coding straight chain maltopentaose generates enzyme The nucleotide sequence of gene is as shown in SEQ ID NO.2.

3. recombined bacillus subtilis according to claim 1 or 2, which is characterized in that be with bacillus subtilis WB600 Expressive host.

4. recombined bacillus subtilis according to claim 1 to 3, which is characterized in that using pST as expression vector.

5. a kind of method for constructing any recombined bacillus subtilis of claim 1-4, which is characterized in that the method packet It includes:

(1) using Bacillus stearothermophilus genomic DNA as template, both ends is obtained by PCR amplification and are contained respectively The g5 target gene fragment of I restriction enzyme site of Nco I and Xho；

(2) by the g5 genetic fragment insertion pMD 18-T simple plasmid obtained in (1), cloning vector pMD 18-T is obtained simple/g51；

(3) using I site Nco inside target gene in rite-directed mutagenesis removal pMD 18-T simple/g51, carrier is obtained pMD 18-T simple/g52；

(4) carrier pMD 18-T simple/g52 recycles the purpose g5 containing cohesive end after I double digestion of Nco I and Xho Genetic fragment is simultaneously inserted into pST plasmid, and pST/g5 plasmid is obtained, and expression vector pST/g5 is converted host bacillus subtilis WB600 obtains genetic engineering bacterium.

6. a kind of method for producing straight chain maltopentaose and generating enzyme, which is characterized in that using any weight of claim 1-4 Group bacillus subtilis is fermented.

7. according to the method described in claim 6, it is characterized in that, fermentation medium used are as follows: yeast powder 30-36g/L, corn Starch 5-7g/L, KH₂PO₄2.0-2.5g/L, K₂HPO₄12-13g/L, pH are 7.0~8.0.

8. according to the method described in claim 6, it is characterized in that, needing before fermentation by seed activation, seed activation includes: Any recombined bacillus subtilis of claim 1-4 is inoculated into LB culture medium, in 35-38 DEG C, 200-220r/ Min cultivates 10-12h.

9. according to the method described in claim 8, it is characterized in that, the seed liquor after activation is turned with the inoculum concentration of 1%-5% It is connected in fermentation medium, in 35-38 DEG C, 200-220r/min cultivates 48-96h.

10. any recombined bacillus subtilis of claim 1-4 prepare maltopentaose or product containing maltopentaose in Application.