CN1495252A

CN1495252A - Prduction method of exoinulase

Info

Publication number: CN1495252A
Application number: CNA021241457A
Authority: CN
Inventors: 王建华; 王明华; 滕达; 张帆; 姚怡; 刘艳艳; 王亚茹
Original assignee: Feed Research Institute of Chinese Academy of Agricultural Sciences
Current assignee: Feed Research Institute of Chinese Academy of Agricultural Sciences
Priority date: 2002-07-12
Filing date: 2002-07-12
Publication date: 2004-05-12

Abstract

The present invention provides a pichia pastoris EXI2086 strain (CGMCC No.0763) which can high-effectively express exoinulase and can be used as production strain. It adopts the high-density fermentation production method to produce exoinulase (maximum exoinulase activity in the fermentation liquor can be up to 577.14 U/ml). The utilization of said invention can raise yield of inulase and can shorten the zymogenic fermentation high-peak stage.

Description

A kind of production method of circumscribed inulinase

The present invention relates to a kind of production method of circumscribed inulinase, belong to the sugar industry field.

Sucrose just is being subjected to the challenge of other more superior product as the dominant position of sweeting agent because of its nutrition drawback.Long-term a large amount of edible sucrose has caused a series of nutrition and health problem---obesity, diabetes, carious tooth, hypertension and cardiovascular disorder etc., and along with this problem of Economic development is outstanding all the more.Therefore: the sucrose substitute R﹠D work is extremely paid attention to.

At present in the world, fructose output rises always and clings to sucrose on one's trail in numerous sucrose substitutes or sweeting agent kind, occupies second.This is because the unique advantage of fructose.Its low heat value becomes sweeting agent and low calorie foods raw material new lover.One of fructose production approach is both at home and abroad at present: be that raw material is the multistep processes approach through three kinds of enzymatic multistep chemical reactions production fructose with starch.With the inulin is that raw material relies on the i.e. industrialization of a step enzyme method production approach of inulin enzyme one-step catalyzed reaction production high purity fructose, is in the starting stage.

The multistep processes explained hereafter high fructose syrups (HFS) that adopt more, be raw material promptly with corn or rice fecula, through 3 kinds of enzyme (amylase, saccharifying enzyme and glucose isomerase) catalyzed reaction and 4～5 reactions steps, the reaction head product be the high fructose syrup that contains fructose 42% (the 1st generation product, F-42), raw material availability and efficiency of pcr product are all very low.By membrane sepn and circulating reaction can prepare contain fructose 55% (the 2nd generation product, F-55) and 90% (the 3rd generation product, high high fructose syrup F-90), but that cost increases is high.One-step technology has characteristics or the advantage different with multistep processes technology: one-step technology has only a step inulinase catalyzed reaction, production process is greatly simplified, production cost reduces, and the saccharification head product is high purity (＞90%) fructose HFS, efficiency of pcr product＞100%; 3 kinds of different enzymic catalytic reactions of multistep processes arts demand and multistep product separating reaction, head product fructose yield＜50%.Obviously, inulinase is the unique important enzyme that a step enzyme method is produced the inulin high fructose syrup.

The general liquid state fermentation production inulinase that adopts.It is reported that a kind of aspergillus niger of inulinase that produces is under the shaking table level, at 30 ℃ of 140r/min, reach 27U/ml to the work of 120h enzyme, the work of 168h enzyme peaks and is 48.4U/ml, and biomass reaches 28.8g/L (OhtaK., Akimoto H., Matsuda., et al., Molecular cloning and sequence analysis of two endo-inulinasegenes from Aspergillus niger.Biosci.Biotechnol.Biochem., 1998,62:1731-1738.).Report (Ongen-Baysal G.and Sukan S.S.Production of inulinase mixed culture of Aspergillius nigerand Kluyveromyces marxianus.Biotechnol.Lett.1996 recently, 18:1431-1434.) a kind of aspergillus niger that produces inulinase shaking under bottle level, 28 ℃ of 200r/min, inulinase reach the peak value of 50U/ml to 168h.The suitableeest growth of one strain K.marxianus is 40～45 ℃ with producing the enzyme temperature, adopts inorganic medium to carry out cultured continuously in the 1L fermentor tank, and culture medium carbon source is restricted to 0.25% sucrose, and fermentation parameter is pO ₂Be controlled at 50%～70%, by 1M KOH and 0.5M H ₂SO ₄Control pH4.5, under the 0.1/h dilution rate, the highest extracellular enzyme 32U/ml alive, I/S=1/15, extracellular enzyme and intracellular enzyme ratio are roughly according to incubation time: 50%～65%/50%～35%, remaining sucrose concentration and production of enzyme are negative correlation (Rouwenhorst R.J. in the substratum, Hensing M., Verbakel J., et al.Structure and properties of the extracellular inulinase of Kluyveromycesmarxianus CBS6556, Appl.Environ.Microbiol., 1988,54:1311-1137; 1990,56:3337-3345.).Another strain K.marxianus NCYC 587 is at 28 ℃ of 200r/min, 120h inulinase activity reach 34.5U/ml (Ongen-BaysalG.and Sukan S.S.Production of inulinase mixed culture of Aspergillius niger andKluyveromyces marxianus.Biotechnology Lett.1996,18:1431-1434.).Another strain Kluyveromyces bacterial strain produces the enzyme orthogonal experiments through shaking bottle on organic substratum of being made up of extractum carnis, corn steep liquor, urea and jerusalem artichoke juice: the active 15U/ml of being of the highest inulinase extracellular enzyme, test gained the highest extracellular enzyme output is respectively 17.63 and 18.7U/ml (Wei Wenling etc. on 15L and 1000L fermentor tank, the research of the synthetic inulinase optimum condition of kluyveromyces Y-85. the microorganism journal, 1998,38:208-212).The optimum carbon source that latest report Bacillus sphaericus produces inulinase is 0.75% inulin, produces inulinase 178.4U/ml; Optimum nitrogen source is an extractum carnis, produce inulinase 196.1U/ml (Kwon S.J, Yoo, J.Y., Lee J.S.Isolation ofintracellular inulinase-producing bacterirum and culture condition for inulinase production.FoodSci.Biotechnol., 1999,8:24-29).Wang Jianhuas etc. adopt yeast high-density cells fermentation process, elected natural bacterial strain Kluyveromyces (CGMCC 0360) is (35～40 ℃ of 120h, 500～800r/min) the highest inulinase output reach 289U/ml, are international production peak enzyme level (Wang Jianhua etc. the nineties in 20th century.A kind of yeast strain of inulinase and the application in high fructose syrup thereof, 1998, Chinese patent, number of patent application No.98120697.2 of producing; Wang Jianhua etc.Inulinase high yield yeast high-density culture and zymologic property research.The biotechnology journal, 2000; 16 (1): 60-64).

Inulinase (Inulinase) is can hydrolysis β-2, a class lytic enzyme of 1-D-Polylevulosan fructose glycosidic bond, formal name used at school β-2, the 1-D-levanase is beta-fructosidase enzyme again, or β-fructan-hydrolying enzyme, or 2,1-D-Polylevulosan fructan-hydrolying enzyme, zymetology is numbered EC 3.2.1.The mode of cutting the Polylevulosan chain according to the inulinase enzyme is divided into restriction endonuclease (EC 3.2.1.7) and excision enzyme (EC3.2.1.80); Early 1930s extracts inulinase from microorganism cells, the further investigation inulinase starts from 20th century the mid-80, and this is that the endoinulase hydrolytic inulin can get high-purity fructo oligosaccharides because circumscribed inulinase hydrolytic inulin can get high purity fructose.Fructose and oligofructose are sweeting agent, functional food ingredient and the additives more more outstanding than sucrose.

The objective of the invention is also this bacterial strain to be used for the method for the suitability for industrialized production of inulinase, to improve inulinase output and to shorten and produce the enzymic fermentation peak period, the problem that inulinase yields poorly, cost is high in the solution production practice for the superior strain of selecting a strain inulinase.

The inventor adopts gene recombination method, obtain the restructuring yeast strains of the circumscribed inulinase of a plant height efficient expression, called after: pichia pastoris phaff reorganization Pichia pastoris EXI2086 bacterial strain, and at China Committee for Culture Collection of Microorganisms common micro-organisms center (No. 13, one in Zhong Guan-cun, BeiJing, China north, 100080) carry out preservation, preserving number is: CGMCCNo.0763, preservation date are on July 10th, 2002.

Another object of the present invention provides a kind of method of being produced circumscribed inulinase by the restructuring yeast strains high density fermentation that the present invention obtained.The method that the inulinase degraded inulin that the tunning that above method is produced utilizes the method for 98120697.2 patent applications that the inventor submitted on January 29th, 1999 to produce is produced high fructose syrup.

Technological line of the present invention is seen accompanying drawing 1.

Specific descriptions of the present invention are as follows:

At first filter out the circumscribed inulinase of a strain and produce bacterial strain, promptly circumscribed inulinase genetic donor bacterial strain is a Kluyveromyces sp Kluyveromyces IW9801 bacterial strain (CGMCC0360); And the character that plays the inulinase that produces studied (seeing 98120697.2 patent applications that the inventor submitted on January 29th, 1999), increase and the preservation plasmid is E.coliTOP10F ' with intestinal bacteria; Design corresponding primer and circumscribed inulinase gene is cloned, again through construction of recombinant vector, acceptor yeast conversion (F-strain: pichia pastoris phaff Pichia pastoris GS115 (His ^-Mut ^-)) and a whole set of gene recombination such as recombinant screen and recon functional assays and express the engineering yeast Pichia pastoris EXI2086 strain that Optimizing operation obtains to efficiently express circumscribed inulinase.

The above work of the present invention obtains:

1. Ke Long circumscribed inulinase full length gene 1599bp (not containing signal peptide), 533 amino acid of encoding, the homology comparative analysis is found: tie up circumscribed inulinase gene order homology＞99% with the external Crewe of report the earliest, regard as same origin gene.Infer that from amino acid theoretical molecular is 59.2KD.10 potential N-glycosylation sites (Asn-X-Ser/Thr, X are arbitrary amino acid) on amino acid sequence coded, have been found.G+C content reaches 47.3% in the circumscribed inulinase gene.

2. successfully make up circumscribed inulinase recombination yeast Pichia pastoris EXI2086, reach high yield enzyme level 577U/ml at 96h; Produce the high yield enzyme level of each bacterial strain that enzyme level is significantly higher than our known domestic and foreign literature report.

The method of being produced inulinase by the restructuring yeast strains high density fermentation that the present invention obtained is described below (unless stated otherwise, the used per-cent of the present invention all is weight percentage):

The engineering strain Pichiapastoris EXI2086 that cultivates the present invention's development in the 6.6L fermentor tank that pH, dissolved oxygen (DO), temperature are controlled automatically produces inulinase.Initial substratum charging volume at fermentor tank is 30% of a tankage: basic medium: 2.67% phosphoric acid, and 0.093% calcium sulfate, 1.82% vitriolate of tartar, 1.49% sal epsom, 0.413% potassium hydroxide, 4.0% glucose, 0.437%PTM1, moisturizing is to 1L.Wherein PTM1 composition and compound concentration are: 0.6% copper sulfate, 0.008% sodium iodide, 0.3% manganous sulfate, 0.002% Sodium orthomolybdate, 0.02% boric acid, 0.05% cobalt chloride, 2.0% zinc chloride, 6.5% ferrous sulfate, 0.025% vitamin H, 0.5% sulfuric acid, moisturizing is to 1L.Engineering strain inoculum size 5%～7.5% (engineering strain shake-flask seed substratum: 1.0% yeast extract, 2% peptone, 2% glucose adds water to 1L), leavening temperature is controlled at 28～32 ℃, and stirring velocity is 800～1,200r/min, pO ₂All be controlled at more than 30%, inlet air flow speed is greater than 9L/min, and the pH of fermented liquid is controlled at (institute's ammoniacal liquor of adding plays the nitrogenous source effect simultaneously in substratum) on the pH5.6 level all the time with 2Mol/l ammoniacal liquor; Produce the inulinase engineering strain and have different control methods, mainly by two portions form (1) by basic medium and growth phase feed supplement liquid (growth phase feed supplement liquid: 25% glucose, 1.2%PTM1, moisturizing is to 1L.When residual sugar in the substratum be lower than 0.5% or biomass carry out feed supplement when being lower than 200g/L) provide enough nutritive substances to obtain more than the maximum biomass 200g/L to quicken the yeast cell breeding; (2) adopt mixing inducing culture (mixing inducing culture (feed supplement liquid): 25% glucose, 10% methyl alcohol, 1.2%PTM1, moisturizing is to 1L) carry out feed supplement, its objective is when further improving biomass, make yeast adapt to the methyl alcohol carbon source, and enter the initial period of inducing exogenous protein to express thus; (3) adopt pure inducing culture (pure inducing culture (feed supplement liquid): pure methyl alcohol, 1.2%PTM1, moisturizing is to 1L) to carry out feed supplement thereafter,, only add the variation of flow at this therebetween, other conditions are constant; The foundation of feed supplement fluctuations in discharge is: keep the DO value on a suitable level, if too low (＜30%) illustrates that growth is fast, oxygen consumption is many, show under-nutrition in the substratum, need to improve the feed supplement flow this moment; If too high (＞40%) illustrates that growth is slow, oxygen consumption is few, show nutritional sufficiency in the substratum, need to lower the feed supplement flow this moment.The condition of abduction delivering and optimization principles and Jayne Stratton et al. method (High Cell-DensityFermentaion.In:David RH., James MC.ed.Methods in Molecular Biology-ProtocolsPichia, 1998, vol.103 Humana Press Inc., New York:107-120) basically identical, just the carbon source glycerine that uses in the substratum is changed, so not only reduced to produce the enzymic fermentation cost but also do not influence and nourished and grown and the exogenous protein expression effect by the glucose replacement.Total incubation time can obtain the inulinase ultimate production of maximum and best product enzyme economic benefit when reaching 90～144h.

The present invention utilizes the nutrient solution of above acquisition,, with after enzyme liquid separates inulinase liquid is mixed according to 1: 8～17 volume ratio with the inulin solution that contains sugar 8～17% through thalline, 45～55 ℃ of following enzymatic saccharifications 30～40 hours, obtains high fructose syrup.The structure of producing bacterial strain comprises following experiment:

Experiment one

The circumscribed inulinase genetic donor of the clone of circumscribed inulinase gene bacterial strain is Kluyveromyces IW9801 (CGMCC0360)

(1) F+strain extracting genome DNA (adopting neutral cracking process) is collected thalline, with distilled water wash once; Thalline is suspended in (1.0M sorbyl alcohol, 0.1M sodium-acetate pH7.0,60mM EDTA in the 0.2ml broken wall system, 1%Zymolyase5000 reaches final concentration 1mg/ml, 1% mercaptoethanol) processing 30min adds following material: 0.5%SDS, 100mM Tris-HClpH7.0 under 37 ℃, 50mM EDTA pH8.5, mixing is put in-70 ℃ of refrigerators, add 0.2ml 5M Potassium ethanoate again, put 0 ℃, 45min, again in 4 ℃ centrifugal 10,000r/min, 10min stays supernatant to be divided into two parts.With the saturated phenol of equal-volume Tris: chloroform: primary isoamyl alcohol (25: 24: 1) extracting once, 15,000r/min, centrifugal 5min collects supernatant, and use the equal-volume chloroform again: primary isoamyl alcohol (24: 1) extracting once, add 2 times of volume precooling dehydrated alcohols, static 30min under room temperature.4 ℃, 10,000r/min, centrifugal 20min abandons supernatant liquor and stays precipitation.Wash twice, 4 ℃ 10 continuously with 1ml precooling 70% ethanol, 000r/min, centrifugal 20min stays precipitation.Precipitation is DNA, is dissolved in 50 μ lTE and preserves standby down in-20 ℃.

(2) circumscribed inulinase gene amplification primer design is a foundation with synthesizing according to the kluyveromyces inulinase complete genome sequence of having reported, 5 ' end at the exoinu gene has 23 amino acid whose signal coding sequences of a coding, method by PCR, with base sequence after the signal coding sequence (long 35 bases) and gene 3 ' terminal sequence (long 29 bases) is primer, with segmentation or disposablely angle that to get goal gene be purpose, the design Auele Specific Primer is some right, and it is synthetic to transfer to Shanghai Genecore Inc, can disposablely successfully angle a pair of primer of getting full goal gene:

P1：5’CAAAGCTTGATTGCGGCCGATGGATGGTGACAGCA?3’

P2：5’CAGAATTCTCAATTTCACCAATAACGTTG?3’

(3) the pcr amplification goal gene is provided with the PCR reaction parameter and is: 94 ℃ of sex change 5min, the back adds the Taq enzyme in reaction system, 6000r/min, centrifugal 20 seconds, circulate 30--35 time the 94 ℃ of sex change 1min that at every turn circulate, 55 ℃ of annealing 1min then, 72 ℃ are extended 1min, 72 ℃ of insulation 10min after the loop ends.The PCR product carries out the low melting-point agarose electrophoresis.

Target gene clone and nucleotide structure analysis

Amplified production low melting-point agarose electrophoresis.Judge that test kit reclaims purified pcr product about amplified fragments size 1.6kb, serve the extra large Genecore biotech company target gene that the clone obtains that checks order, obtain this gene nucleotide series after measured, the result is as follows:

5’GATGGTGACA?GCAAGGCCAT?CACTAACACC?ACTTTTAGTT?TGAACAGACC?TTCTGTGCAT 60

TTCACTCCAT?CCCATGGTTG?GATGAACGAT?CCAAATGGTT?TGTGGTACGA?TGCCAAGGAA 120

GAAGACTGGC?ATTTGTACTA?CCAGTACAAC?CCAGCAGCCA?CGATCTGGGG?TACTCCATTG 180

TACTGGGGTC?ACGCTGTTTC?CAAGGATTTG?ACTTCCTGGA?CAGATTACGG?TGCTTCTTTG 240

GGCCCAGGTT?CCGACGACGC?TGGTGCGTTC?AGTGGTAGTA?TGGTTATCGA?TTATAACAAT 300

ACTTCTGGTT?TCTTCAACAG?CTCTGTGGAC?CCAAGACAAA?GAGCAGTTGC?AGTCTGGACT 360

TTGTCTAAGG?GCCCAAGCCA?AGCCCAACAC?ATCAGTTACT?CATTGGACGG?TGGTTACACC 420

TTCGAGCACT?ACACCGACAA?CGCCGTGTTG?GACATCAACA?GCTCCAACTT?CAGAGACCCT 480

AAGGTGTTCT?GGCACGAGGG?CGAGAACGGC?GAAGATGGTC?GTTGGATCAT?GGCCGTTGCT 540

GAATCGCAAG?TGTTCTCTTG?TTTGTTCTAC?TCTTCTCCAA?ACTTGAAAAA?CTGGACCTTG 600

GAATCCAACT?TCACCCACCA?CGGCTGGACT?GGTACCCAAT?ACGAATGTCC?AGGTCTAGTT 660

AAGGTTCCAT?ACGACAGTGT?TGTTGACTCT?TCGAACTCCT?CCGACTCCAA?GCCAGACTCC 720

GCATGGGTCT?TGTTTGTCTC?TATCAACCCT?GGTGGTCCAT?TGGGTGGTTC?CGTTACCCAA 780

TACTTTGTTG?GTGACTTCAA?CGGTACTCAC?TTCACTCCAA?TCGACGGCCA?AACCAGATTC 840

CTAGACATGG?GTAAGGACTA?CTACGCACTA?CAAACTTTCT?TCAACACTCC?AAACGAGAAG 900

GACGTCTACG?GTATCGCATG?GGCTTCTAAC?TGGCAATACG?CCCAACAAGC?CCCAACTGAC 960

CCATGGCGTT?CATCTATGAG?TTTGGTTAGA?CAATTCACAT?TGAAAGACTT?CAGCACAAAC 1020

CCTAACTCCG?CTGATGTCGT?CTTGAACAGT?CAACCAGTCT?TGAACTATGA?TGCATTGAGA 1080

AAGAACGGTA?CCACTTACAG?TATCACAAAC?TACACCGTCA?CCTCCGAAAA?CGGCAAGAAG 1140

ATCAAGCTAG?ACAACCCATC?CGGTTCTCTT?GAATTCCATC?TTGAATACGT?GTTTAACGGC 1200

TCCCCAGATA?TCAAGAGCAA?CGTGTTCGCT?GATCTTTCCT?TGTACTTCAA?GGGTAACAAC 1260

GACGACAACG?AATACTTGAG?ATTGGGTTAC?GAAACCAACG?GTGGTGCCTT?CTTCTTGGAC 1320

CGTGGCCACA?CCAAGATTCC?TTTCGTGAAG?GAGAACTTGT?TCTTCACCCA?CCAATTGGCA 1380

GTTACCAACC?CAGTTTCCAA?CTACACCACA?AACGTCTTCG?ACGTTTACGG?TGTCATTGAC 1440

AAGAACATCA?TCGAATTGTA?CTTCGATAAC?GGTAACGTCG?TCTCCACCAA?CACTTTCTTC 1500

TTCTCTACCA?ACAACGTTAT?TGGTGAAATT?GACATCAAGT?CGCCATACGA?CAACCCTTAC 1560

ACCATTAACT?CATTTAACGT?TACCCAATTT?AACGTTTGA?3’ 1599

Aminoacid sequence by Nucleotide decryptography is as follows:

GAT?GGT?GAC?AGC?AAG?GCC?ATC?ACT?AAC?ACC?ACT?TTT?AGT?TTG?AAC?AGA?CCT?TCT 54

D G D S K A I T N T T F S L N R P S 18

GTG?CAT?TTC?ACT?CCA?TCC?CAT?GGT?TGG?ATG?AAC?GAT?CCA?AAT?GGT?TTG?TGG?TAC 108

V H F T P S H G W M N D P N G L W Y 36

GAT?GCC?AAG?GAA?GAA?GAC?TGG?CAT?TTG?TAC?TAC?CAG?TAC?AAC?CCA?GCA?GCC?ACG 162

D A K E E D W H L Y Y Q Y N P A A T 54

ATC?TGG?GGT?ACT?CCA?TTG?TAC?TGG?GGT?CAC?GCT?GTT?TCC?AAG?GAT?TTG?ACT?TCC 216

I W G T P L Y W G H A V S K D L T S 72

TGG?ACA?GAT?TAC?GGT?GCT?TCT?TTG?GGC?CCA?GGT?TCC?GAC?GAC?GCT?GGT?GCG?TTC 270

W T D Y G A S L G P G S D D A G A F 90

AGT?GGT?AGT?ATG?GTT?ATC?GAT?TAT?AAC?AAT?ACT?TCT?GGT?TTC?TTC?AAC?AGC?TCT 324

S G S M V I D Y N N T S G F F N S S 108

GTG?GAC?CCA?AGA?CAA?AGA?GCA?GTT?GCA?GTC?TGG?ACT?TTG?TCT?AAG?GGC?CCA?AGC 378

V D P R Q R A V A V W T L S K G P S 126

CAA?GCC?CAA?CAC?ATC?AGT?TAC?TCA?TTG?GAC?GGT?GGT?TAC?ACC?TTC?GAG?CAC?TAC 432

Q A Q H I S Y S L D G G Y T F E H Y 144

ACC?GAC?AAC?GCC?GTG?TTG?GAC?ATC?AAC?AGC?TCC?AAC?TTC?AGA?GAC?CCT?AAG?GTG 486

T D N A V L D I N S S N F R D P K V 162

TTC?TGG?CAC?GAG?GGC?GAG?AAC?GGC?GAA?GAT?GGT?CGT?TGG?ATC?ATG?GCC?GTT?GCT 540

F W H E G E N G E D G R W I M A V A 180

GAA?TCG?CAA?GTG?TTC?TCT?TGT?TTG?TTC?TAC?TCT?TCT?CCA?AAC?TTG?AAA?AAC?TGG 594

E S Q V F S C L F Y S S P N L K N W 198

ACC?TTG?GAA?TCC?AAC?TTC?ACC?CAC?CAC?GGC?TGG?ACT?GGT?ACC?CAA?TAC?GAA?TGT 648

T L E S N F T H H G W T G T Q Y E C 216

CCA?GGT?CTA?GTT?AAG?GTT?CCA?TAC?GAC?AGT?GTT?GTT?GAC?TCT?TCG?AAC?TCC?TCC 702

P G L V K V P Y D S V V D S S N S S 234

GAC?TCC?AAG?CCA?GAC?TCC?GCA?TGG?GTC?TTG?TTT?GTC?TCT?ATC?AAC?CCT?GGT?GGT 756

D S K P D S A W V L F V S I N P G G 252

CCA?TTG?GGT?GGT?TCC?GTT?ACC?CAA?TAC?TTT?GTT?GGT?GAC?TTC?AAC?GGT?ACT?CAC 810

P L G G S V T Q Y F V G D F N G T H 270

TTC?ACT?CCA?ATC?GAC?GGC?CAA?ACC?AGA?TTC?CTA?GAC?ATG?GGT?AAG?GAC?TAC?TAC 864

F T P I D G Q T R F L D M G K D Y Y 288

GCA?CTA?CAA?ACT?TTC?TTC?AAC?ACT?CCA?AAC?GAG?AAG?GAC?GTC?TAC?GGT?ATC?GCA 918

A L Q T F F N T P N E K D V Y G I A 306

TGG?GCT?TCT?AAC?TGG?CAA?TAC?GCC?CAA?CAA?GCC?CCA?ACT?GAC?CCA?TGG?CGT?TCA 972

W A S N W Q Y A Q Q A P T D P W R S 324

TCT?ATG?AGT?TTG?GTT?AGA?CAA?TTC?ACA?TTG?AAA?GAC?TTC?AGC?ACA?AAC?CCT?AAC 1026

S M S L V R Q F T L K D F S T N P N 342

TCC?GCT?GAT?GTC?GTC?TTG?AAC?AGT?CAA?CCA?GTC?TTG?AAC?TAT?GAT?GCA?TTG?AGA 1080

S A D V V L N S Q P V L N Y D A L R 360

AAG?AAC?GGT?ACC?ACT?TAC?AGT?ATC?ACA?AAC?TAC?ACC?GTC?ACC?TCC?GAA?AAC?GGC 1134

K N G T T Y S I T N Y T V T S E N G 378

AAG?AAG?ATC?AAG?CTA?GAC?AAC?CCA?TCC?GGT?TCT?CTT?GAA?TTC?CAT?CTT?GAA?TAC 1188

K K I K L D N P S G S L E F H L E Y 396

GTG?TTT?AAC?GGC?TCC?CCA?GAT?ATC?AAG?AGC?AAC?GTG?TTC?GCT?GAT?CTT?TCC?TTG 1242

V F N G S P D I K S N V F A D L S L 414

TAC?TTC?AAG?GGT?AAC?AAC?GAC?GAC?AAC?GAA?TAC?TTG?AGA?TTG?GGT?TAC?GAA?ACC 1296

V F N G S P D I K S N V F A D L S L 432

AAC?GGT?GGT?GCC?TTC?TTC?TTG?GAC?CGT?GGC?CAC?ACC?AAG?ATT?CCT?TTC?GTG?AAG 1350

N G G A F F L D R G H T K I P F V K 450

GAG?AAC?TTG?TTC?TTC?ACC?CAC?CAA?TTG?GCA?GTT?ACC?AAC?CCA?GTT?TCC?AAC?TAC 1404

E N L F F T H Q L A V T N P V S N Y 468

ACC?ACA?AAC?GTC?TTC?GAC?GTT?TAC?GGT?GTC?ATT?GAC?AAG?AAC?ATC?ATC?GAA?TTG 1458

T T N V F D V Y G V I D K N I I E L 486

TAC?TTC?GAT?AAC?GGT?AAC?GTC?GTC?TCC?ACC?AAC?ACT?TTC?TTC?TTC?TCT?ACC?AAC 1512

V F D N G N V V S T N T F F F S T N 504

AAC?GTT?ATT?GGT?GAA?ATT?GAC?ATC?AAG?TCG?CCA?TAC?GAC?AAC?CCT?TAC?ACC?ATT 1566

N V I G E I D I K S P Y D N P Y T T 522

AAC?TCA?TTT?AAC?GTT?ACC?CAA?TTT?AAC?GTT?TGA 1599

N S F N V T Q F N V Z 533

Analyze the nucleotide sequence and the amino acid sequence coded of circumscribed inulinase gene, have following feature:

(1) Ke Long 1599 Nucleotide of circumscribed inulinase gene (not containing signal peptide) total length, 533 amino acid of encoding, the homology comparative analysis is found: tie up circumscribed inulinase gene order homology＞99% (Laloux O. with the external Crewe of report the earliest, J-P.Cassart, J.Delcour, et al., 1991), assert that the two has same origin gene.

(2) infer that from amino acid theoretical molecular is 59.2KDa.

(3) found 10 potential N-glycosylation sites (Asn-X-Ser/Thr, X are arbitrary amino acid) on amino acid sequence coded, the proteic molecular weight of maturing enzyme is 78KDa.

(4) this gene G+C content 47.3%.

Experiment two

With Pichia pastoris GS115 is the construction of recombinant vector of recipient bacterium

With EcoRI and HindIII respectively enzyme cut exoinu gene and pUC18, carry out the low melting-point agarose electrophoresis after enzyme is cut and reclaim the required fragment of purifying, exoinu and pUC18 under the effect of T4DNA ligase enzyme, spend the night in 16 ℃ after (molar ratio) mixed with 3: 1 ratios.Get ligation thing transformed competence colibacillus E.coli JM103, go up screening recon pUC18A at the LB of 37 ℃ of incubated overnight solid medium (containing Amp 50～100 μ g/ml), extract plasmid, enzyme is cut evaluation.Downcut the fragment that comprises exoinu from the pUC18A of empirical tests with EcoRI and SnaBI enzyme, cut pPIC9 with EcoRI and SnaBI enzyme equally, carry out the low melting-point agarose electrophoresis after enzyme is cut and reclaim the required fragment of purifying, exoinu and pPIC9 are with 3～5: after 1 (molar ratio) mixes, under the effect of T4DNA ligase enzyme, spend the night in 16 ℃.Get ligation thing transformed competence colibacillus E.coli TOP10F ' then, be coated with flat board on LB substratum (containing Amp50～100 μ g/ml), 37 ℃ of incubated overnight are selected positive recombinant.PCR verifies recombinant plasmid, extract plasmid, plasmid with extraction is Template DNA, with 5 ' AOX1 sequencing primer:5 '-GACTGGTTCCAATTGACAAGC-3 ' and 3 ' AOX1 sequencingprimer:5 '-GCAAATGGCATTCTGACATCC-3 ' is primer, carries out PCR reaction and angles and get characteristic dna fragmentation (seeing accompanying drawing 2).

Verify recombinant vectors with PCR, sequencing result shows that (pPIC9 length is 8023bp with correct reading frame and Yeast expression carrier pPIC9 by EcoRI and SnaBI site for exoinu gene through transforming, have the Amp resistance marker, have alcohol oxidase enzyme promoter sequence 3 ' AOX1 (1-948bp) and alpha--excreted factor signal peptide sequence (948-1218bp), has multiple clone site XhoI, SnaBI, EcoRI, NotI etc. are between 1192-1241bp.) on alpha-factor signal coding sequence 3 ' end merge, obtain recombinant vectors pPIC9A.Foreign gene exoinu gene on the recombinant vectors is subjected to the control of AOX1 (alcohol oxidase enzyme 1) promotor like this.

Experiment three

Yeast conversion and positive recombinant screening

F-strain is pichia pastoris phaff Pichia pastoris GS115 (His ^-Mut ^-), 30 ℃ are cultured to A in 500ml YPD (1.0% yeast extract, 2% peptone, 2% glucose) ₆₀₀=1.3～1.8 o'clock centrifugal collection thalline; Use the aseptic washing thalline of 500ml and 250ml precooling successively, the centrifugal supernatant that goes is collected thalline; 1mol/L sorbyl alcohol suspension thalline with the 20ml precooling; Use the sorbyl alcohol suspension thalline of 0.5ml precooling after centrifugal again; Get 40 μ l, add 1～5 μ g linearizing recombinant plasmid, ice bath 5min transforms parameter: 0.8kV, 11.5 μ F with LN-101 electric shock instrument electric shock.The 1mol/L sorbyl alcohol that adds the 0.5ml precooling after electric shock finishes is immediately got 200 μ l and is gone up coated plate in solid YSDB, and 30 ℃ of cultivations occur until transformant.On MM and MD flat board, cultivate 2d down for 30 ℃, the clone's (his undesired or that do not grow that on MM, grows with the dibbling of the correspondence of the transformant on the aseptic toothpick picking YSDB growing normal on the MD ⁺Mut ^-) positive clone's.

Transformant can be gone up growth at substratum YSDB (not containing His), but not transformant can not grow, and this is because recipient bacterium is histidine defect type (his ^-), though and the His4 gene is arranged on the carrier, do not have the yeast replicon, so the His4 gene on the carrier must be integrated in the yeast genes group and could express.In addition, because the AOX1 gene is damaged in the yeast cell of reorganization, so it can not utilize methyl alcohol as carbon source again, like this with methyl alcohol as the substratum of sole carbon source on transformant just can not grow or grow very slow, show as methyl alcohol and utilize defective type (Mut ^-).

Experiment four

The Molecular Identification of recombination yeast

Recombination yeast is 30 ℃ of violent joltings in 10ml BMGY, make cell grow to state of saturation, and centrifugal collection thalline adds 10ml inducing culture BMMY, and 30 ℃ were continued inducing culture 5～7 days down, after recombination yeast is cultivated, extract genomic dna in BMGY.Carry out the PCR reaction, with the recombination yeast genomic dna as dna profiling, with 5 ' AOX1sequencing primer 5 '-GACTGGTTCCAATTGACAAGC-3 ' and 3 ' AOX1 sequencing primer:5 '-GCAAATGGCATTCTGACATCC-3 ' is that primer carries out pcr amplification, whether correctly inserts expression vector to prove circumscribed inulinase gene.Send the PCR product order-checking of outside Shanghai Genecore biotech company, prove that circumscribed inulinase gene correctly inserts in the genomic dna.

Experiment five

Expression of recombinant yeast product alantin excision enzyme SDS-PAGE analyzes

The centrifugal thalline that goes behind 30 ℃ of inducing culture 36h of recombination yeast, get 3 μ l supernatant liquors and carry out the SDS-PAGE analysis, (acrylamide: methylene diacrylamide is 29: 1) resolving gel concentration is 8%, concentrated gum concentration is 5%, after electrophoresis finishes, gel then decolours with 10% glacial acetic acid with the Coomassie brilliant blue 30min that dyes.

Protein s DS-PAGE electrophoresis result shows that the circumscribed inulinase molecular weight size of expression is about 78KDa, after handling with Endo H (Endo-β-N-acetylglyco-saminidase H) de-glycosylation, molecular weight is reduced to about 60KDa, proved that from protein level recon can normal expression and the circumscribed inulinase protein of secretion external source, expression product can carry out glycosylation modified behind the protein translation.

Following experiment material is used in experiment one to experiment five

The circumscribed inulinase genetic donor of 1 bacterial strain and plasmid bacterial strain is that Kluyveromyces IW9801 (CGMCC0360), amplification and preservation plasmid are E.coli DH5 α with intestinal bacteria, E.coli TOP10F ', F-strain Pichia pastorisGS115 (His ^-Mut ^-), plasmid pUC18 and pPIC9 are feed biotechnology research chamber Wang Jianhua seminar of Institute of Feeds,China Academy of Agriculture Sciences and Yao Bin seminar preserves and provide material.5 ' AOX1 sequencing primer:5 '-GACTGGTTCCAATTGACAAGC-3 ' and 3 ' AOX1 sequencing primer:5 '-GCAAATGGCATTCTGACATCC-3 ' primer are the Invitrogen product; Other materials is common research proprietary concentrate.

2 enzymes and test kit are glad from Beijing to be bought through reagent company of section and Promega company.Comprise: restriction enzyme EcoRI, HindIII, SnaBI, the TaqDNA polysaccharase, the T4DNA ligase enzyme, RNase, universal PC R reaction kit, extracting genome DNA and purification kit, dna fragmentation reclaims and purification kit.

3 substratum

(1) intestinal bacteria substratum: 1% peptone, 0.5% yeast extract, 1%NaCl, pH7.0.

(2) perfect medium YPD:1.0% yeast extract, 2% peptone, 2% glucose.

(3) transform substratum YSDB:1.34%YNB, 18.6% sorbyl alcohol, 2% glucose, 0.00004%Biotin, 0.005% L-glutamic acid, 0.005% methionine(Met), 0.005% Methionin, 0.005% leucine, 0.005% Isoleucine, 2% agar.

(4) select substratum MM:1.34%YNB, 0.00004%Biotin, 0.5% methyl alcohol, 2.0% agar.

(5) select (contrast) substratum MD:1.34%YNB, 0.00004%Biotin, 2% glucose, 2.0% agar.

(6) induce (adaptation) substratum BMGY:1% yeast extract, 2% peptone, 1.34%YNB, 0.00004%Biotin, 1% glycerine is regulated medium pH 6.0 by the 0.1Mol/l sodium phosphate buffer.

(7) inducing culture BMMY:1% yeast extract, 2% peptone, 1.34%YNB, 0.00004%Biotin, 0.5% methyl alcohol is regulated medium pH 6.0 by the 0.1Mol/l sodium phosphate buffer.

Experiment six

Recombination yeast Pichia pastoris EXI2086 expresses circumscribed inulinase research

Random choose is 120 from 200 positive transformants, be inoculated in finger-type test tube and 15 milliliters of centrifuge tubes that the BMGY substratum is housed, at 250r/min, cultivate 24h down for 30 ℃, centrifugal then conversion BMMY substratum directly detects the inulinase activity of inductive fermented liquid, according to active size behind the 3d, selected some strain subcultures select the last 6.6 liters of Braun experiment fermentor tanks of 1 high yield recon (name and be EXI2086) (Biostat) research condition of enzyme production and curve more than 10 generations.Main fermentation process and Jayne Stratton et al. method (In:David RH., James MC.ed., Methods in MolecularBiology-Protocols Pichia, 1998, vol.103 Humana Press Inc., New York:107-120.) basic identical, coefficient 0.30 is for the later stage produces the enzyme induction feed supplement and gives and stop enough spaces.

1 substratum

(1) engineering strain shake-flask seed substratum: 1.0% yeast extract, 2% peptone, 2% glucose adds water to 1L.

(2) fermentor tank basic medium composition: 2.67% phosphoric acid, 0.093% calcium sulfate, 1.82% vitriolate of tartar, 1.49% sal epsom, 0.413% potassium hydroxide, (preceding 5 kinds of compositions can be prepared and become, 5～10 times of concentrated mother liquors), 4% glucose, 0.437%PTM1 solution, defoamer GP 0.2ml, GPE 0.8ml, moisturizing to 1 liter.

(3) micro-PTM1 composition: 0.6% copper sulfate, 0.08% sodium iodide, 0.3% sulfuric acid is violent, 0.02% Sodium orthomolybdate, 0.02% boric acid, 0.05% cobalt chloride, 2% zinc chloride, 6.5% ferrous sulfate, 0.025% vitamin H, 0.5% vitriol oil, moisturizing to 1 liter.

(4) growth phase feed supplement liquid composition: 25% glucose contains PTM1 composition 12ml/l (feed supplement liquid).

(5) feed supplement liquid composition is induced in mixing: 25% glucose, and 10% methyl alcohol contains PTM1 composition 12ml/l (feed supplement liquid);

(6) pure inducing culture: pure methyl alcohol contains PTM1 composition 12ml/l (feed supplement liquid).

The 6.6L Braun fermentor tank (Biostat that 2 fermentation conditions are controlled automatically in pH, DO, temperature, Germany's product) culturing engineering bacterial strain Pichia pastoris EXI2086 produces circumscribed inulinase in, engineering strain is 2.0L at the initial charging volume of fermentor tank, inoculum size 5%～7.5%, leavening temperature is controlled at 30 ℃ respectively, stirring velocity is 1,200r/min, pO ₂All be controlled at more than 30%, inlet air flow speed is greater than 9L/min, and the pH of fermented liquid is controlled at pH5.6 (institute's ammoniacal liquor of adding plays the nitrogenous source effect simultaneously in substratum) respectively with 2Mol/l ammoniacal liquor; Produce circumscribed inulinase engineering strain and have different control methods, mainly forming (1) by three parts provides enough nutritive substances to obtain maximum biomass to quicken the yeast cell breeding by initial medium and supplemented medium, (2) supplementary feeding target protein induced expression material and nutritive substance mixed culture are when further improving biomass, begin to induce the expression of target protein, the condition of abduction delivering and Jayne Stratton et al. method (High Cell-Density Fermentation, In:David RH., James MC.ed.Methods in MolecularBiology-Protocols Pichia, 1998, vol.103 Humana Press Inc., New York:107-120) basically identical, just the carbon source glycerine that uses in the substratum is changed by glucose and replace, so not only reduced cost but also do not influence the effect of nourishing and growing.Other variation comprises that also growth medium replenishes the regulation and control of material and induced expression make-up stream speed, being described in detail among the embodiment 1 that concrete parameter is seen below.

Table 1. recombination yeast Pichia pastoris EXI2086 produces circumscribed inulinase time course

Time (h) biomass (through 5000g, 10min is centrifugal for g/l, FW) unit of enzyme activity (U/ml)

0 10 --

24 98 --

30 165 --

36 180 60.34

48 198 104.16

56 210 141.19

60 230 300.95

72 230 314.76

84 250 393.80

96 254 577.14

Table 1 is as seen: the high yield inulinase level of Pichia pastoris EXI2086 reaches 577.14U/ml at 96h, higher 1 times than original donor starting strain Kluyveromyces marxianus IW9801 (CGMCC0360) highest level (289U/ml), and the time shortens 24h, express the characteristics of other heterologous proteins according to this yeast expression system, also have enough optimization leeway, expection can reach better product enzyme level.

Experiment seven

The active measuring method of circumscribed inulinase

This research inulinase reaction conditions is: enzyme liquid 0.50ml and 2.5%～3.0% synanthrin (Shanghai chemical reagent two factories) 0.50ml through suitably dilution react 10min at 55 ℃; reaction system is 1ml; pH value of reaction system is 4.5, is provided by 0.02mmol/L acetate-sodium acetate buffer.By reducing sugar content in the Fehlings reagent assaying reaction system.Inulinase activity unit is defined as--and to produce the required enzyme amount of 1 μ mol hexose be 1 unit of enzyme activity to every milliliter of per minute in the reaction system.

Description of drawings:

Fig. 1 technological line of the present invention

Fig. 2. the circumscribed inulinase recombination yeast of recombinant vectors pPIC9A and high expression level Pichia pastoris EXI2086 makes up

The present invention has following characteristics: (1) engineered strain Pichia pastoris EXI2086 produces enzyme level 577.14U/ml, height The similar research of finding in the inventor or the peak of patent report, on March 4th, 2002 was looked into new website through country-level science and technology Retrieval shows (200101c1400033): this is unique exoinulinase worker with successful structure of production meaning up to now The journey bacterial strain; (2) reaching the high yield enzyme level time shortens to 96h from the 120h of starting strain, in advance 24h; (3) obtain The needed biomass of same enzyme level is lower than starting strain; (4) 30 ℃ of engineered strain fermentation temperatures are lower than starting strain 8 ℃; (5) replace the appointment carbon source glycerine of this expression system with glucose, front 5 all mean the fermentation energy and cost Decrease; (6) producing exoinulinase and inducing proper proportion and the medium pH of glucose and methyl alcohol between culture period Control is to optimize one of 2 important factors of condition of enzyme production; (7) be unique high yield that produces exoinulinase in reporting at present Bacterial strain, the purifying cost of saving enzyme.

Embodiment 1

The seed liquor preparation: inclined-plane seed (1 ring) inserts substratum (shake-flask seed substratum (YPD): 1% yeast culture, 2% peptone, 2% glucose, H is housed ₂O 1000ml) 250ml shakes in the bottle through 30 ℃ of 24h, and 300r/min becomes seed liquor, changes the 500ml that same substratum is housed again over to and shakes in the bottle through 30 ℃ of 24h, and 300r/min becomes secondary seed solution.

Growth phase: the preparation of 2.0L medium base material (is contained 2.67% phosphoric acid, 0.093% calcium sulfate, 1.82% vitriolate of tartar, 1.49% sal epsom, 0.413% potassium hydroxide, 4.0% glucose, 0.437%PTM1 (PTM1 composition: 0.6% copper sulfate, 0.08% sodium iodide, 0.3% manganous sulfate, 0.02% Sodium orthomolybdate, 0.02% boric acid, 0.05% cobalt chloride, 2.0% zinc chloride, 6.5% ferrous sulfate, 0.025% vitamin H, 0.5% vitriol oil) defoamer GP 0.2ml/L, GPE 0.8ml/L, the 6.6L fermentor tank pack into through 121 ℃ of steam sterilizing 30min, be cooled to 30 ℃ and insert seed liquor, 30 ℃ of cultivations, 1,200r/min stirs, and the pH of fermented liquid is controlled at (institute's ammoniacal liquor of adding plays the nitrogenous source effect simultaneously in substratum) on the pH5.6 level, pO all the time with 2Mol/L ammoniacal liquor ₂〉=30%.After cultivating 24h, press 30 ℃ of the speed feed supplements (feed supplement liquid composition: 25% glucose contains PTM1 composition 12ml/L) of 36ml/h/L, pO ₂〉=40%, behind the 5h, ventilate with 9L/min; 1,200r/min stirs, pH5.6 (replenishing the ammoniacal liquor regulation and control automatically).

Induction period: carry out feed supplement with mixing inducing culture (25% glucose, 10% methyl alcohol contain PTM1 composition 12ml/L) to 48h, its flow is: 1h is 36ml/h/L, and the 2nd to 4h is 19.2ml/h/l, and other conditions are the same.Again the back with the speed of 3ml/h/L with inducing feed supplement liquid to carry out feed supplement (feed supplement liquid composition: 10% pure methyl alcohol contains PTM1 composition 12ml/L), pO purely ₂＞=50%, other conditions are the same.Use with the speed of 6ml/h/L again behind the feed supplement 12h and mix inducing culture (25% glucose, 10% methyl alcohol contain PTM1 composition 12ml/L) feed supplement 6h again, pO ₂Be 50%～55%, other conditions are the same.With inducing feed supplement liquid to carry out feed supplement purely, other conditions are the same to finish cultivation to 96h with the speed of 12～10ml/h/L in the back again.Product enzyme level in the fermented liquid reaches 577.14U/ml at this moment.

Claims

1. the circumscribed inulinase of a strain produces bacterial strain, it is characterized by this bacterial strain is that the inventor adopts molecular biological method, a strain engineering strain that obtains, called after: Pichia pastoris EXI2086, and carry out preservation at China Committee for Culture Collection of Microorganisms common micro-organisms center, preserving number is: CGMCC No.0763, preservation date is: on July 10th, 2002.

2. produce bacterial strain according to the described circumscribed inulinase of claim 1, it is characterized by this bacterial strain obtains by the following method, that is: at first filter out the circumscribed inulinase of a strain and produce bacterial strain, promptly circumscribed inulinase genetic donor bacterial strain is KluyveromycesIW9801, CGMCC No.0360); Through amplification, design corresponding primer, and circumscribed inulinase gene is cloned, again through construction of recombinant vector, be transformed into F-strain Pichia yeast Pichia pastoris GS115 (His ^-Mut ^-) in, again the Yeast engineering bacterium strain that obtains to efficiently express circumscribed inulinase is identified, screened to the recon process that obtains.

3. the production method of a circumscribed inulinase is characterized in that the engineering strain Pichia pastoris EXI2086 that cultivates the present invention's development in the 6.6L fermentor tank that pH, dissolved oxygen, temperature are controlled automatically produces inulinase.Initial basic medium charging volume at fermentor tank is 30% of a tankage: engineering strain inoculum size 5%～7.5%, leavening temperature are controlled at 28～32 ℃, and stirring velocity is 800～1,200r/min, pO ₂All be controlled at more than 30%, inlet air flow speed is greater than 9L/min, and the pH of fermented liquid is controlled on the pH5.6 level all the time with 2Mol/l ammoniacal liquor, and institute's ammoniacal liquor of adding plays the nitrogenous source effect simultaneously in substratum; Produce circumscribed inulinase engineering strain and have different control methods, mainly forming (1) by two portions provides enough nutritive substances to obtain maximum biomass and reach 200g/L at least to quicken the yeast cell breeding by basic medium and growth phase feed supplement liquid, when residual sugar in the substratum be lower than 0.5% or biomass carry out feed supplement when being lower than 200g/L; (2) adopt the mixing inducing culture to carry out feed supplement, its objective is when further improving biomass, make recombination microzyme adapt to the methyl alcohol carbon source, and enter the initial period of inducing exogenous protein to express thus; (3) adopt pure inducing culture to carry out feed supplement thereafter,, only add the variation of flow at this therebetween, other conditions are constant; The foundation of feed supplement fluctuations in discharge is: keep dissolved oxygen on a suitable level, if＜30%, illustrate that growth is fast, oxygen consumption is many, show under-nutrition in the substratum, need to improve the feed supplement flow this moment; If＞40%, illustrate that growth is slow, oxygen consumption is few, show nutritional sufficiency in the substratum, need to lower the feed supplement flow this moment.The condition of abduction delivering and optimization principles and Jayne Stratton et al. method basically identical, just the carbon source glycerine that uses in the substratum is changed, so not only reduced to produce the enzymic fermentation cost but also do not influence and nourished and grown and the exogenous protein expression effect by the glucose replacement.Total incubation time can obtain the inulinase ultimate production of maximum and best product enzyme economic benefit when reaching 90～144h.

The present invention utilizes the nutrient solution of above acquisition,, with after enzyme liquid separates inulinase liquid is mixed according to 1: 8～17 volume ratio with the inulin solution that contains sugar 8～17% through thalline, 45～55 ℃ of following enzymatic saccharifications 30～40 hours, obtains high fructose syrup.

4. the production method of the described circumscribed inulinase of claim 3 is characterized in that used basic medium is: 2.67% phosphoric acid, 0.093% calcium sulfate, 1.82% vitriolate of tartar, 1.49% sal epsom, 0.413% potassium hydroxide, 4.0% glucose, 0.437%PTM1, moisturizing is to 1L.Wherein PTM1 composition and compound concentration are: 0.6% copper sulfate, 0.008% sodium iodide, 0.3% manganous sulfate, 0.002% Sodium orthomolybdate, 0.02% boric acid, 0.05% cobalt chloride, 2.0% zinc chloride, 6.5% ferrous sulfate, 0.025% vitamin H, 0.5% sulfuric acid, moisturizing is to 1L.

5. according to the production method of the described circumscribed inulinase of claim 3, it is characterized in that used engineering strain shake-flask seed substratum: 1.0% yeast extract, 2% peptone, 2% glucose adds water to 1L.

6. according to the production method of the described circumscribed inulinase of claim 3, it is characterized in that used growth phase feed supplement liquid is: 25% glucose, 1.2%PTM1, moisturizing is to 1L.

7. according to the production method of the described circumscribed inulinase of claim 3, it is characterized in that used mixing inducing culture is: 25% glucose, 10% methyl alcohol, 1.2%PTM1, moisturizing is to 1L.

8. according to the production method of the described circumscribed inulinase of claim 3, it is characterized in that used pure inducing culture is: pure methyl alcohol, 1.2%PTM1, moisturizing is to 1L.

9. according to the production method of the described circumscribed inulinase of claim 3, it is characterized in that the highest circumscribed inulinase activity reaches 577.14U/ml in the fermented liquid.