CN102071171B - Bifunctional glutathione synthetase and method for producing glutathione by using same - Google Patents

Abstract

The invention discloses a method for producing glutathione by using bifunctional glutathione synthetase contained in actinobacillus pleuropneumonia, actinobacillus succinogenes, bacillus cereus, streptococcus sanguis, streptococcus gordonii, streptococcus uberis and streptococcus thermophilus. The method comprises heterologous expression by using the seven microbes or enzymes of the microbes in other microbes. The glutathione synthesized by using the method has the structure of natural glutathione, and has high response rate and high output and yield of the glutathione.

Description

A kind of difunctional glutathione synthetase and utilize it to produce the method for gsh

Technical field

The present invention relates to a kind of method of producing gsh, concretely, is to utilize a kind ofly to have the gamma-glutamylcysteine synthetic enzyme (γ-GCS) and the enzyme of two kinds of functions of glutathione synthetase (GS) are produced the method for gsh.

Background technology

Gsh (Glutathione, be called for short GSH) be a kind of compound that contains sulfydryl with important physiological function, the tripeptides that is formed by Pidolidone (L-Glu), Cys (L-Cys) and three amino acid of glycine (Gly).Gsh is distributed widely in animal, plant, the microorganism, participates in the synthetic of protein and Yeast Nucleic Acid in organism, the transportation of oxygen and nutritive substance, keep endogenous enzyme activity, tricarboxylic acid cycle and carbohydrate metabolism in the body are removed functions such as crossing polyradical in the body, have widely and use.Clinically, gsh be used for radioprotective, antitumor, anticancer, antioxygen poison, anti-ageing and coordinate endocrine treatment.As a kind of antioxidant, gsh also is used as foodstuff additive, is used for prolonging the quality guaranteed period of food and the aminoacid component in the nutrient fortified food.

The main production method of gsh has extraction method, chemical synthesis, fermentation method and enzyme process.

Extraction method (introducing such as United States Patent (USP) 3396033) is take the abundant animal and plant of glutathione content and yeast as raw material, by adding suitable solvent or in conjunction with the processing of amylase, proteolytic enzyme, separating obtaining through multistep again.Because the content of natural tissues GSH-PX activity is low, only be 0.5～1.0% of dry weight, and treatment step is many, so that the overall yield of the method is not high, so extraction method is substituted by other method gradually.

The gsh that chemical synthesis is produced is the raceme of D-type and L-type, and wherein, what have physiologically active is GSH, so, expect that activated gsh need to carry out optical resolution with the product of chemosynthesis.In addition, the step of chemical synthesis is various, and the time is longer, pollutes large.

In recent years, more to the report with Glutathione Production by Microbial Fermentation, the level of fermentation method is also improving gradually.Synthesizing glutathion needs two-step reaction in the organism: the first step is that Pidolidone and Cys are at Mg ²⁺And ATP exists, and lower (effect of γ-GCS) generates γ-L-glutamyl-Cys by the gamma-glutamylcysteine synthetic enzyme; Second step is that gamma-L-glutamine-cysteine and glycine are at Mg ²⁺And ATP exists lower effect by glutathione synthetase (GS) to generate γ-GSH, i.e. gsh.Two-step reaction all needs the participation of ATP.(γ-GCS) is subject to the feedback inhibition of reduced glutathion to first enzyme of synthesizing glutathion in most organisms, and in the production process of gsh, the feedback inhibition that solves product is to improve the key of gsh output.

At present, the research of much being devoted to make up the GSH superior strain being arranged, is to want the γ-GCS and the GS overexpression in Host Strains that utilize engineered method will derive from intestinal bacteria or yeast saccharomyces cerevisiae, thereby reaches the purpose of GSH high yield.Such as: γ-GCS and the GS of Chen Jian etc. (Letters in Applied Microbiology (2006) 43:211-214) in intestinal bacteria in the overexpression intestinal bacteria, in the situation of two copies of the gene gsh I of coding γ-GCS and a copy of coding GS gene gsh II, the GSH production level of recombinant bacterium JM109 (pVB03) can reach 34.8mg g ^-1Wet thallus; Chen Shaoxin etc. (Bioprocess Biosystem Engineering (2009) 32:729-735) express γ-GCS and the GS in the yeast saccharomyces cerevisiae in pichia spp, recombinant bacterium Pichia pastoris GS115 (pGAPZHGSH) carries out high density fermentation, cell concentration can reach 98.15g (dry weight)/L, and the concentration of contained GSH reaches 4.15g/L.These concentration of studying thalline main GSH content from the raising thalline or the raising fermenting process reach the purpose of GSH high yield.It is by protein engineering releasing GSH the feedback inhibition angle of γ-GCS to be set out to improve GSH output that research is also arranged.

The people such as Griffith find, the enzyme that a kind of γ of having-GCS and two kinds of functions of GS are arranged in streptococcus agalactiae (Streptococcus agalactiae), called after γ-GCS-GS, it is 32% identical that the N-terminal sequence of this enzyme and colibacillary γ-GCS have, 43% similar, its C-terminal sequence and the GS that reported be without any similarity, but with D-alanine-D-alanine ligase enzyme have 24% identical, 38% similar.Derive from γ-GCS-GS of streptococcus agalactiae to the feedback insensitive of GSH, and GSH do not have restraining effect to the activity of γ-GCS and GS, this organism glutathion inside synthetic enzyme from previous report is different.The people such as Aharonowitz have also reported a kind of enzyme of the same race that is present in the Listeria Monocytogenes (Listeria monocytogenes) subsequently, and called after GshF, its N-terminal sequence is similar to γ-GCS, the GS of its C-terminal sequence and report does not have obvious homology, and is similar to ATP in conjunction with albumen.Also there is same enzyme in the discoveries such as Van Beeumen Pasteurella multocida (Pasteurella multocida), GSH is little to the more colibacillary γ-GCS of feedback inhibition of the GshF in Pasteurella multocida source, GSH is 13.6mM to the inhibition constant K i of GshF, and GSH is about 3.mM to the Ki of colibacillary γ-GCS, and GSH is about 140mM to the Ki of γ-GCS-GS of streptococcus agalactiae.

Summary of the invention

The object of the present invention is to provide by some microorganisms, have gamma-glutamylcysteine synthetic enzyme (γ-GCS) and a glutathione synthetase of two kinds of functions of glutathione synthetase (GS) simultaneously.

Second purpose of the present invention is to provide a kind of described method of being produced gsh by the difunctional glutathione synthetase of microorganisms of utilizing.

The 3rd purpose of the present invention is to provide a kind of method of utilizing described microbial enzyme to produce gsh at other microorganism heterogenous expressions.

For achieving the above object, the present invention realizes by the following technical programs one by one:

A kind of difunctional glutathione synthetase (is continued to use forefathers' name; be referred to as GshF); have simultaneously two kinds of functions of gamma-glutamylcysteine synthetic enzyme and glutathione synthetase, described difunctional glutathione synthetase is by one or more generations in the following microorganism:

Actinobacillus pleuropneumoniae (Actinobacillus pleuropneumonia), Actinobacillus succinogenes (Actinobacillus succinogenes), bacillus cereus (Bacillus cereus), Streptococcus sanguis (Streptococcus sanguinis), Gall's chain coccus (Streptococcusgordonii), streptococcus uberis (Streptococcus uberis), thermophilus streptococcus (Streptococcusthermophilus).

The aminoacid sequence of described difunctional glutathione synthetase is respectively such as SEQ ID NO:1---shown in the SEQ IDNO:7.

Described difunctional glutathione synthetase is encoded by gshF, and its gene order is respectively such as SEQ ID NO:8---shown in the SEQ ID NO:14.

A kind of method of utilizing difunctional glutathione synthetase synthesizing glutathion is the catalyzer synthesizing glutathion by actinobacillus pleuropneumoniae, Actinobacillus succinogenes, bacillus cereus, Streptococcus sanguis, Gall's chain coccus, streptococcus uberis and thermophilus streptococcus; Or be the catalyzer synthesizing glutathion by the difunctional glutathione synthetase that utilizes these microorganisms to extract.

A kind of method of utilizing difunctional glutathione synthetase synthesizing glutathion is the catalyzer synthesizing glutathion by the genetic engineering bacterium of expressing actinobacillus pleuropneumoniae, Actinobacillus succinogenes, bacillus cereus, Streptococcus sanguis, Gall's chain coccus, streptococcus uberis and thermophilus streptococcus gshF; Or the difunctional glutathione synthetase that is extracted by the genetic engineering bacterium of expressing actinobacillus pleuropneumoniae, Actinobacillus succinogenes, bacillus cereus, Streptococcus sanguis, Gall's chain coccus, streptococcus uberis and thermophilus streptococcus gshF is the catalyzer synthesizing glutathion.

A kind of method of utilizing difunctional glutathione synthetase synthesizing glutathion is produced by the genetic engineering bacterium fermentation of cultivating actinobacillus pleuropneumoniae, Actinobacillus succinogenes, bacillus cereus, Streptococcus sanguis, Gall's chain coccus, streptococcus uberis and thermophilus streptococcus or culture expression actinobacillus pleuropneumoniae, Actinobacillus succinogenes, bacillus cereus, Streptococcus sanguis, Gall's chain coccus, streptococcus uberis and thermophilus streptococcus gshF.

Utilize described difunctional glutathione synthetase, with the precusor amino acids synthesizing glutathion, this difunctional glutathione synthetase is produced by wild mushroom or genetic engineering bacterium; The wild mushroom cell of the described difunctional glutathione synthetase of utilization expression or genetically engineered mycetocyte are as catalyzer, with the precusor amino acids synthesizing glutathion; The wild mushroom of the described difunctional glutathione synthetase of culture expression or genetic engineering bacterium glutathion production by fermentation.The gene of described difunctional glutathione synthetase can require 1 described microorganism amplification to obtain by the PCR Accessory Right, also can obtain by full gene is synthetic, can replace the codon of non-preference according to host's codon preference.

The synthetic GSH method that the present invention proposes can directly be utilized the above-mentioned wild mushroom that contains difunctional GSH synthetic enzyme, also can by round pcr with the gshF gene amplification in vitro in the bacterial strain of above several different sourcess after, be connected in the expression vector, these recombinant vectorss are converted into heterogenous expression GshF albumen in the recipient bacterium.Various microorganisms be used to carrying out genetic expression all can be used as the recipient bacterium of overexpression gshF gene, comprise bacterium (such as intestinal bacteria, Bacillus subtillis, pseudomonas etc.), yeast (such as yeast saccharomyces cerevisiae, pichia pastoris phaff, multiple-shaped nuohan inferior yeast etc.), mould (such as aspergillus oryzae, aspergillus niger etc.).Collect the recombinant bacterium cell of wild mushroom or overexpression GshF, pass through penetratingization processing as catalyzer, or separate its GshF as catalyzer, add again and contain three kinds of precusor amino acids and Mg ²⁺Damping fluid in, add ATP or utilize cell itself or other microbial metabolism glucose produce ATP, biocatalysis precusor amino acids Pidolidone, Cys and glycine generation gsh.

These GshF genes can adopt conventional genetic engineering means to obtain, as obtaining by PCR take above-mentioned strain chromosome DNA as template, or synthetic according to its sequence.When adopting synthetic method, can according to the preference of different expression systems to codon, the codon of host's preference will be replaced with for the rare codon of host partly or entirely in these GshF genes of coding.Replace to the codon of intestinal bacteria preference such as the leucic codon CTA that will wherein encode when the expression in escherichia coli, coding arginic codon AGG, CGA; Also can be with intestinal bacteria rare codon AGG all in the sequence, CGA, CTA, ATA, CCC, TGT, TGC, GGA, GGG replace to the codon of intestinal bacteria preference.

Said gshF gene can be introduced host cell by expression vector independently duplicated in host cell, also can be by being incorporated on the recipient bacterium karyomit(e) such as methods such as homology restructuring.Usually being used for the Host Strains that carries out genetic expression can comprise bacterium as the recipient bacterium of overexpression GshF, such as intestinal bacteria, Bacillus subtillis, pseudomonas, actinomycetes etc.; Yeast is such as yeast saccharomyces cerevisiae, pichia pastoris phaff, multiple-shaped nuohan inferior yeast etc.; Mould comprises aspergillus oryzae, aspergillus niger etc.The expression of gshF gene can be adopted general efficient inducible promoter, constitutive promoter, such as P _L, lac, trc, gap, AOX1, MOX, gpd, the promotors such as gla, amy, also can adopt the promotor of gshF gene self.

Containing the wild mushroom of difunctional GSH synthetic enzyme or the recombinant bacterium of overexpression gshF gene can adopt conventional method to cultivate, as adopt the fed batch cultivation method to carry out high-density culture, the somatic cells that acquisition has high GshF activity in a large number is as the biological catalyst of synthetic GSH.Enter the efficient that cell is participated in the glutathione synthesis reaction in order to improve substrate, the cell of the above-mentioned GshF of containing activity can carry out penetratingization processing, and synthetic GSH mainly is present in the reaction solution, is convenient to its separation and purification.Penetratingization processing can be adopted ordinary method, as thalline is freezed cell in-20 ℃, keep certain hour after, in 37 ℃ of thawings, one to three time repeatedly; Or carry out penetratingization processing with toluene etc.Also the GshF that recombinant bacterium or wild mushroom are expressed can be separated, with the GshF after separating as biological catalyst or immobilization after as catalyzer, synthetic GSH.The wild mushroom with GshF activity of above-mentioned penetratingization or the GshF biological catalyst of recombinant bacterium cell or separation carry out the synthetic of GSH in the damping fluid that contains Pidolidone, Cys and three kinds of precusor amino acids of glycine.Synthesize in the reaction of GSH at the GshF with full cell or separation, reaction solution is 0.01-.8mol/L potassium phosphate buffer (pH7.0-8.0), adds Mg ²⁺5-30mmol/L, ATP10-120mmol/L, and precusor amino acids Pidolidone 5-80mmol/L, Cys 5-60mmol/L, glycine 5-80mmol/L was 28-42 ℃ of reaction 2-10 hour.During take somatic cells as catalyzer, the wet thallus add-on is 5-25% (w/v).Also can not add ATP, metabolic activity by cell self is with glucose degradation and produce ATP, or the metabolic reaction coupling that the glucose degradation that adds in the reaction solution is produced ATP with other cells (such as yeast saccharomyces cerevisiae), provide synthetic GSH reaction required ATP, the amount that glucose adds is 0.1-1.0mol/L.

The invention has the beneficial effects as follows this production level that can obviously be improved gsh by the difunctional glutathione synthetase of described seven kinds of microorganisms; Utilize the recombinant microorganism of the difunctional glutathione synthetase of the described microorganism of overexpression to produce gsh, production level more is improved largely.

Description of drawings

Fig. 1 is the PCR product electrophorogram of actinobacillus pleuropneumoniae gshF;

Fig. 2 is the structure of recombinant plasmid pTrc99A-gshFap;

Fig. 3 is the structure of recombinant plasmid pET28a-gshFap;

Fig. 4 is the structure of recombinant plasmid pYES2-gshFap;

Fig. 5 is the PCR product electrophorogram of thermophilus streptococcus gshF;

Fig. 6 is the PCR product electrophorogram of Actinobacillus succinogenes gshF;

Fig. 7 is the PCR product electrophorogram of bacillus cereus gshF;

Fig. 8 is the PCR product electrophorogram of Streptococcus sanguis gshF;

Fig. 9 is the PCR product electrophorogram of Gall's chain coccus gshF;

Embodiment

Below in conjunction with accompanying drawing and 14 embodiment to further instruction of the present invention.

Experiment material and working method brief description:

1, enzyme and main agents

Used biomaterial comprises bacterial strain actinobacillus pleuropneumoniae with gshF gene and thermophilus streptococcus etc., e. coli jm109, e. coli bl21 (DE3), bacillus coli DH 5 alpha, plasmid pTrc99A, pET28A, yeast saccharomyces cerevisiae INVSc1, plasmid pYES2.Restriction enzyme EcoR I, Hind III, NotI, T4 ligase enzyme etc. are available from Takara company.Genome extraction agent box, dna gel reclaim test kit, the plasmid extraction test kit is purchased from Shanghai and gives birth to worker company.

2, molecular biology operation

PCR reaction conditions, intestinal bacteria and yeast saccharomyces cerevisiae competence are made and method for transformation reference " molecular cloning experiment guide ".The purifying of the extraction of genomic dna, plasmid extraction, DNA, enzyme are cut and are connected all and operate by product description.

Embodiment 1 utilizes the Actinobacillus succinogenes synthesizing glutathion

1, the cultivation of Actinobacillus succinogenes (Actinobacillus succinogenes)

Actinobacillus succinogenes 130Z (CICC 11014) is pressed 1% inoculum size access fermention medium from the glycerine pipe of-20 ℃ of preservations, and concrete composition is (containing among the 1L): wood sugar 5g, yeast extract 5g, corn steep liquor 5g, Na ₂HPO ₄12H ₂O 0.5g, NaH ₂PO ₄2H ₂O 0.5g, 6.5,37 ℃ of pH, 220rpm, shaking culture 24 hours.

2, cell pretreatment

Cultured Actinobacillus succinogenes nutrient solution at the centrifugal 5min collecting cell of 8000rpm, is used 0.05mol/L, and the phosphoric acid buffer of pH7.0 washs centrifugal three times under same condition.Cell in-20 ℃ freezing 2 hours, carry out penetratingization processing.

3, glutathione synthesis reaction

The 2g wet thallus that takes by weighing penetratingization processing adds in the 20mL reaction solution, and reaction solution is 0.2mol/L (pH7.0) potassium phosphate buffer, contains Pidolidone 40mmol/L, Cys 20mmol/L, glycine 40mmol/L, MgCl ₂6H ₂O 20mmol/L, ATP 40mmol/L, in 37 ℃ of reactions 8 hours, glutathione concentrations can reach 343.7mg/L; E. coli jm109 glutathione synthesis amount is 63.6mg/L under the same terms.

Embodiment 2 utilizes the bacillus cereus synthesizing glutathion

With Bacillus cereus (CGMCC 1.932) from the glycerine pipe of-20 ℃ of preservations by 1% inoculum size access fermention medium, concrete composition is (containing among the 1L): Tryptones 10g, yeast extract 5g, NaCl10g, 37 ℃, 220rpm, shaking culture 12 hours, by embodiment 1 described method bacillus cereus is carried out penetratingization processing and glutathione synthesis reaction, reacted 8 hours, gsh can accumulate 163mg/L.

The structure of embodiment 3 recombination bacillus coli JM109 (pTrc99a-gshFap)

1, the extraction of actinobacillus pleuropneumoniae Actinobacillus pleuropneumonia genomic dna

With Actinobacillus pleuropneumonia (CVCC 259) from the glycerine pipe of-20 ℃ of preservations by 1% inoculum size access fermention medium, concrete composition is (containing among the 1L): peptone 10.0g, yeast extract 5g, NaCl 10g, NAD 0.2g, pH 7.4,37 ℃, leave standstill and cultivate after 24 hours, in 13400 * g, 4 ℃ centrifugal 2 minutes, collect thalline.Adopt BBI complete genome DNA extraction agent box to extract Actinobacillus pleuropneumonia complete genome DNA.

2, actinobacillus pleuropneumoniae gshF gene clone

Upstream primer is: 5 '-GCGC GGATCCATGAAATTACAACAAC-3 ', underscore partly are BamH I restriction enzyme sites.

Downstream primer is 5 '-GAT GTCGACTTAAGGCAGTTCTGGGAA-3 ', underscore partly are Sal I restriction enzyme sites.

Take the full genome of Actinobacillus pleuropneumonia as template, with above primer PCR amplification gshF gene.

The PCR condition is: unwind in advance 94 ℃, 5min; Unwind 94 ℃, 1min anneals 52 ℃, and 1min extends 72 ℃, 3min, cycle number 30; 72 ℃ are extended 10min.Obtain length and be the dna segment about 2.3kb.PCR product electrophorogram as shown in Figure 1.The pig pleuropneumonia unwrapping wire bar gshF gene order sequencing result that the present invention uses is shown in SEQ ID NO:8, and the aminoacid sequence AA1 of the GshF of the actinobacillus pleuropneumoniae of coding sees SEQ ID NO:1.

The gshF that derives from pig pleuropneumonia unwrapping wire bar CVCC 259 has submitted NCBI to, numbering GU138097.

3, the structure of recombinant expression plasmid pTrc99A-gshFap

The gshF gene of pcr amplification and expression vector pTrc99A cut in 37 ℃ of enzymes with restriction enzyme BamH I, SalI spend the night, reclaim respectively the purpose band after the agarose electrophoresis, two fragments are under the effect of T4 ligase enzyme, and 16 ℃ of ligations 12 hours obtain the pTrc99A-gshFap plasmid.Building process as shown in Figure 2.

4, the conversion of the competent making of e. coli jm109 and recombinant plasmid

E. coli jm109 is received in the LB substratum (containing Tryptones 10g/L among the 1L, yeast extract 5g/L, NaCl 10g/L) 37 ℃, the 220rpm incubated overnight, get 30 μ l and be seeded to 3ml LB substratum, 37 ℃, the 220rpm cultivation reached between the 0.4-0.6 OD in about 2 hours, culture is placed 1.5ml eppendorf pipe, behind the ice bath 20min in 4 ℃, 5000rpm, centrifugal 5min, abandon supernatant, add the CaCl of the 100mmol/L of 0.75ml precooling ₂, with washing after the thalline suspension, again in 4 ℃, 5000rpm, centrifugal 5min abandons supernatant, and same method is washed once again, uses at last 100 μ l, the CaCl of 100mmol/L ₂Thalline is suspended, put for subsequent use on ice.

The recombinant plasmid pTrc99A-gshFap that 10 μ l are connected adds in the 100 μ l competent cells, ice bath 30 minutes, heat shock is 90 seconds in 42 ℃ of water-baths, rear rapidly placement on ice, the LB substratum that adds 1mL after 5 minutes, in 37 ℃ of recoveries 1 hour, with the recovery after culture in 3000rpm, centrifugal 10 minutes, take out the wherein supernatant liquor of 1mL, the LB that remaining nutrient solution is coated the amicillin resistance that contains 100mg/L is dull and stereotyped, cultivates the screening recon for 37 ℃, take out plasmid enzyme restriction checking and order-checking, show that pTrc99A-gshFap successfully is converted into e. coli jm109.

The structure of embodiment 4 recombination bacillus coli BL21 (pET28a-gshFap)

PCR product and plasmid pET28a BamH I and Sal with actinobacillus pleuropneumoniae gshF

I cuts at 37 ℃ of enzymes and spends the night; Reclaim the purpose band after the agarose electrophoresis, with the fragment of gshF gene and pET28a under the effect of T4 ligase enzyme, 16 ℃ are spent the night, get pET28a-gshFap.Building process as shown in Figure 3.Press the method for transformation of embodiment 3, change recombinant plasmid pET28a-gshFap over to e. coli bl21, select recon at the kantlex flat board that contains 10mg/L.Extract plasmid, cutting checking and check order by enzyme proves that gshF correctly inserts.

The structure of embodiment 5 recombinant Saccharomyces cerevisiae INVSc (pYES2-gshFap)

Recombinant plasmid pET28a-gshFap and plasmid pYES2 cut at 37 ℃ of enzymes with BamH I and Not I spend the night, cut glue after the agarose electrophoresis and reclaim, with the fragment of gshF gene and pYES2 under the effect of T4DNA ligase enzyme, 16 ℃ are spent the night, get plasmid pYES2-gshFap, its building process as shown in Figure 4.Method with recombinant plasmid pYES2-gshFap presses embodiment 1 changes bacillus coli DH 5 alpha over to, selects recon at the penbritin flat board that contains 100mg/L.The plasmid of being with cut by enzyme and check order the proof plasmid be correct recombinant plasmid pYES2-gshFap.

The recombinant shuttle plasmid pYES2-gshFap of intestinal bacteria and yeast saccharomyces cerevisiae changes uracil auxotrophy yeast saccharomyces cerevisiae INVSc1 over to by the electric shock conversion method, at SC-UD nutrient agar (YNB 6.7g/L, glucose 20g/L, adenosine 0.1g/L, arginine 0.1g/L, halfcystine 0.1g/L, leucine 0.1g/L, Threonine 0.1g/L, tryptophane 0.1g/L, 0.1g/L, aspartic acid 0.05g/L, Histidine 0.05g/L, Isoleucine 0.05g/L, methionine(Met) 0.05g/L, phenylalanine 0.05g/L, proline(Pro) 0.05g/L, Serine 0.05g/L, tyrosine 0.05g/L, α-amino-isovaleric acid 0.05g/L, agar 20g/L) upper screening recon.Method validation with bacterium colony PCR.

The structure of embodiment 6 recombination bacillus coli JM109 (pTrc99a-gshFst)

1, the extraction of thermophilus streptococcus Streptococcus thermophilus genomic dna

Streptococcus thermophilus (SIIM B218) is pressed 1% inoculum size access fermention medium from the semisolid medium (containing the 1L: skim-milk 100g, 20% murphy juice 100mL, yeast extract paste 5g) of 4 ℃ of preservations, and concrete composition is (containing among the 1L): casein 10g, beef extract 10g, yeast extract 5g, glucose 5g, sodium acetate 5g, dibasic ammonium citrate 2g, Tween 801g, K ₂HPO ₄2g, MgSO ₄7H ₂O 0.2g, MnSO ₄H ₂O 0.05g, CaCO ₃6.8,37 ℃ of 20g, pH, the 220rpm shaking culture is after 12 hours, in 13400 * g, 4 ℃ centrifugal 2 minutes, collect thalline.Press the same method extracting Streptococcus thermophilus complete genome DNA of embodiment 3.

2, thermophilus streptococcus gshF gene clone

Upstream primer is: 5 '-CGGC GAATTCACGATGACATTAAACCA-3 ', underscore partly are EcoR I restriction enzyme sites.

Downstream primer is 5 '-GGC AAGCTTTTAAGTTT GACCAGCCACT-3 ', underscore partly are Hind III restriction enzyme sites.

Take the full genome of Streptococcus thermophilus as template, with above primer PCR amplification gshF gene.

The PCR condition is: unwind in advance 94 ℃, 5min; Unwind 94 ℃, 1min anneals 52 ℃, and 1min extends 72 ℃, 3min, cycle number 30; 72 ℃ are extended 10min.Obtain length and be the DNA about 2.3kb.PCR product electrophorogram as shown in Figure 5.The thermophilus streptococcus gshF gene order sequencing result that the present invention uses is shown in SEQ ID NO:14, and the aminoacid sequence AA7 of the GshF of coding sees SEQ ID NO:7.

The gshF that derives from thermophilus streptococcus SIIM B218 has submitted NCBI to, numbering GU138096.

3, the structure of recombinant expression plasmid pTrc99A-gshFst

The thermophilus streptococcus gshF gene of pcr amplification and expression vector pTrc99A cut in 37 ℃ of enzymes with restriction enzyme EcoR I, Hind III spend the night, reclaim respectively the purpose band after the agarose electrophoresis, two fragments are under the effect of T4 ligase enzyme, 16 ℃ of ligations 12 hours obtain the pTrc99A-gshFst plasmid.Prove that by extracting plasmid enzyme restriction checking and order-checking gshF correctly inserts.By embodiment 3 methods pTrc99A-gshFst is transformed JM109 and get JM109 (pTrc99A-gshFst).

The structure of embodiment 7 recombination bacillus coli BL21 (pET28-gshFst)

Plasmid pTrc99A-gshFst and plasmid pET28a cut at 37 ℃ of enzymes with EcoR I and Hind III spend the night; Cut glue after the agarose electrophoresis and reclaim, with the fragment of gshF gene and pET28a under the effect of T4DNA ligase enzyme, 16 ℃ are spent the night, get pET28a-gshFst.Press the method for transformation of embodiment 3, change recombinant plasmid pET28a-gshFst over to e. coli bl21, select recon at the kantlex flat board that contains 10mg/L.Prove that by extracting plasmid enzyme restriction checking and order-checking gshF correctly inserts.

The structure of embodiment 8 recombinant Saccharomyces cerevisiae INVSc (pYES2-gshFst)

Recombinant plasmid pET28a-gshFst and plasmid pYES2 cut at 37 ℃ of enzymes with Hind III and Not I spend the night, cutting glue after the agarose electrophoresis reclaims, with with the fragment of gshF gene and pYES2 under the effect of T4DNA ligase enzyme, 16 ℃ are spent the night, and get plasmid pYES2-gshFst.Method with recombinant plasmid pYES2-gshF presses embodiment 3 changes bacillus coli DH 5 alpha over to, selects recon at the penbritin flat board that contains 100mg/L.Prove that by extracting plasmid enzyme restriction checking and order-checking plasmid is correct recombinant plasmid.

Change recombinant plasmid pYES2-gshFst over to yeast saccharomyces cerevisiae INVSc1 by the method for embodiment 5 again, and select recon.Insertion with the method validation gshF of bacterium colony PCR.

The structure of embodiment 9 recombination bacillus coli BL21 (pET28a-gshFas)

1, the extraction of Actinobacillus succinogenes Actinobacillus succinogenes genomic dna

Actinobacillus succinogenes 130Z (CICC 11014) is pressed 1% inoculum size access fermention medium from the glycerine pipe of-20 ℃ of preservations, and concrete composition is (containing among the 1L): wood sugar 5g, yeast extract 5g, corn steep liquor 5g, Na ₂HPO ₄12H ₂O 0.5g, NaH ₂PO ₄2H ₂O 0.5g, 6.5,37 ℃ of pH, 220rpm, shaking culture is after 24 hours, in 13400 * g, 4 ℃ centrifugal 2 minutes, collect thalline.Adopt BBI complete genome DNA extraction agent box to extract Actinobacillus succinogenes complete genome DNA.

2, Actinobacillus succinogenes gshF gene clone

Upstream primer is: 5 '-GCG GAGCTCATGAATTTACGGCATATA-3 ', underscore partly are Sac I restriction enzyme sites.

Downstream primer is 5 '-GCG CTCGAGTCATGCATTTAATAATTCC-3 ', underscore partly are Xho I restriction enzyme sites.

Take the full genome of Actinobacillus succinogenes 130Z as template, with above primer PCR amplification gshF gene fragment.

The PCR condition is: unwind in advance 94 ℃, 5min; Unwind 94 ℃, 1min anneals 52 ℃, and 1min extends 72 ℃, 3min, cycle number 30; 72 ℃ are extended 10min.Obtain length and be the DNA about 2.3kb.PCR product electrophorogram as shown in Figure 6.The Actinobacillus succinogenes gshF gene order sequencing result that the present invention uses is shown in SEQ ID NO:9, identical with the gshF gene order of the Actinobacillus succinogenes130Z that reports among the NCBI, the aminoacid sequence AA2 of the GshF of coding sees SEQ ID NO:2.

3, the structure of recombinant expression plasmid pET28a-gshFas

Actinobacillus succinogenes gshF gene and expression vector pET28a restriction enzyme Sac I with pcr amplification, Xho I cuts in 37 ℃ of enzymes and spends the night, reclaim respectively the purpose band after the agarose electrophoresis, two fragments are under the effect of T4 ligase enzyme, 16 ℃ of ligations 12 hours obtain the pET28a-gshFas plasmid.Prove that by extracting plasmid enzyme restriction checking and order-checking gshF correctly inserts.

Method with recombinant plasmid pET28a-gshFas presses embodiment 3 is converted into e. coli bl21, namely obtains recombinant bacterium BL21 (pET28a-gshFas).

The structure of embodiment 10 other source gshF recombinant bacteriums

1, the structure of recombination bacillus coli BL21 (pET28a-gshFbc)

(1) extraction of bacillus cereus Bacillus cereus genomic dna

With Bacillus cereus (CGMCC 1.932) from the glycerine pipe of-20 ℃ of preservations by 1% inoculum size access fermention medium, concrete composition is (containing among the 1L): peptone 10g, yeast extract 5g, NaCl10g, 37 ℃, 220rpm, after the shaking culture 12 hours, in 13400 * g, 4 ℃ centrifugal 2 minutes, collect thalline.Adopt BBI complete genome DNA extraction agent box to extract Bacillus cereus complete genome DNA.

(2) bacillus cereus gshF gene clone

Upstream primer is: 5 '-GCG GGATCCATGGAAATGAAAAAAATG-3 ', underscore partly are BamH I restriction enzyme sites.

Downstream primer is 5 '-GGGCGG AAGCTTTTATTTTTTTGGTAG-3 ', underscore partly are Hind III restriction enzyme sites.

Take the full genome of Bacillus cereus as template, with above primer PCR amplification gshF gene.

The PCR condition is: unwind in advance 94 ℃, 5min; Unwind 94 ℃, 1min anneals 52 ℃, and 1min extends 72 ℃, 3min, cycle number 30; 72 ℃ are extended 10min.Obtain length and be the DNA about 2.3kb.PCR product electrophorogram as shown in Figure 7.The bacillus cereus gshF gene order that the present invention uses is shown in SEQID NO:10, and the aminoacid sequence AA3 of the GshF of coding sees SEQ ID NO:3.

(3) structure of recombinant expression plasmid pET28a-gshFbc and conversion

GshF gene and expression vector pET28a restriction enzyme BamH I with pcr amplification, Hind III cuts in 37 ℃ of enzymes and spends the night, and cuts respectively band after the agarose electrophoresis and reclaims, and two fragments are under the effect of T4 ligase enzyme, 16 ℃ of ligations 12 hours obtain the pET28a-gshFbc plasmid.Method with recombinant plasmid pET28a-gshFbc presses embodiment 3 is converted into e. coli bl21, namely obtains recombinant bacterium BL21 (pET28a-gshFbc).Prove that by extracting plasmid enzyme restriction checking and order-checking gshF correctly inserts.

2, the structure of recombination bacillus coli BL21 (pET28a-gshFss)

(1) extraction of Streptococcus sanguis Streptococcus sanguinis genomic dna

With Streptococcus sanguinis (CGMCC 1.2497) from the glycerine pipe of-20 ℃ of preservations by 1% inoculum size access fermention medium, concrete composition is (containing among the 1L): heart extract 5.0g, brain extract 10g, show peptone 10g, glucose 2g, NaCl 5g, Na ₂HPO ₄2.5g 7.4,37 ℃ of pH leave standstill and cultivate after 24 hours, in 13400 * g, 4 ℃ centrifugal 2 minutes, collect thalline.Adopt BBI complete genome DNA extraction agent box to extract Streptococcus sanguinis complete genome DNA.

(2) Streptococcus sanguis gshF gene clone

Upstream primer is: 5 '-GCGA GGATCCATGATGACGATTAATC-3 ', underscore partly are BamH I restriction enzyme sites.

Downstream primer is 5 '-GTAC AAGCTTTCAGTCCATTTCTGGGA-3 ', underscore partly are Hind III restriction enzyme sites.

Take the full genome of Streptococcus sanguinis as template, with above primer PCR amplification gshF gene.

The PCR condition is: unwind in advance 94 ℃, 5min; Unwind 94 ℃, 1min anneals 52 ℃, and 1min extends 72 ℃, 3min, cycle number 30; 72 ℃ are extended 10min.Obtain length and be the DNA about 2.3kb.PCR product electrophorogram as shown in Figure 8.The Streptococcus sanguis gshF gene order NA4 that the present invention uses is shown in SEQID NO:11, and the aminoacid sequence AA4 of the GshF of coding sees SEQ ID NO:4.

(3) structure of recombinant expression plasmid pET28a-gshFss and conversion

The gshF gene of pcr amplification and expression vector pET28a cut in 37 ℃ of enzymes with restriction enzyme BamH I, HindIII spend the night, cutting respectively band after the agarose electrophoresis reclaims, two fragments are under the effect of T4 ligase enzyme, and 16 ℃ of ligations 12 hours obtain the pET28a-gshFss plasmid.The method of pressing embodiment 3 with recombinant plasmid transformed to e. coli bl21, by extracting the plasmid enzyme restriction checking and order-checking proof gshF correctly inserts.

3, the structure of recombination bacillus coli BL21 (pET28a-gshFag)

(1) extraction of Gall's chain coccus Streptococcus gordonii genomic dna

With Streptococcus gordonii (CGMCC 1.2496) from the glycerine pipe of-20 ℃ of preservations by 1% inoculum size access fermention medium, concrete composition is (containing among the 1L): heart extract 5.0g, brain extract 10g, show peptone 10g, glucose 2g, NaCl 5g, Na ₂HPO ₄2.5g 7.4,37 ℃ of pH leave standstill and cultivate after 24 hours, in 13400 * g, 4 ℃ centrifugal 2 minutes, collect thalline.Adopt BBI complete genome DNA extraction agent box to extract Streptococcus gordonii complete genome DNA.

(2) Gall's chain coccus gshF gene clone

Upstream primer is: 5 '-GCG GGATCCATGACGATTAATCAAT-3 ', underscore partly are BamH

The I restriction enzyme site.

Downstream primer is 5 '-GTT GTCGACTTACAGGTCCATTTCTG-3 ', underscore partly are Sal

The I restriction enzyme site.

Take the full genome of Streptococcus gordonii as template, with above primer PCR amplification gshF gene.

The PCR condition is: unwind in advance 94 ℃, 5min; Unwind 94 ℃, 1min anneals 52 ℃, and 1min extends 72 ℃, 3min, cycle number 30; 72 ℃ are extended 10min.Obtain length and be the DNA about 2.3kb.PCR product electrophorogram as shown in Figure 9.The Gall's chain coccus gshF gene order that the present invention uses is shown in SEQ IDNO:12, and the aminoacid sequence AA5 of the GshF of coding sees SEQ ID NO:5.

(3) structure of recombinant expression plasmid pET28a-gshFsg and conversion

GshF gene and expression vector pET28a restriction enzyme BamH I with pcr amplification, SalI cuts in 37 ℃ of enzymes and spends the night, and reclaims respectively the purpose segment after the agarose electrophoresis, and two fragments are under the effect of T4 ligase enzyme, 16 ℃ of ligations 12 hours obtain the pET28a-gshFsg plasmid.Method with recombinant plasmid pET28a-gshFsg presses embodiment 3 is converted into e. coli bl21, namely obtains recombinant bacterium BL21 (pET28a-gshFsg).Prove that by extracting plasmid enzyme restriction checking and order-checking gshF correctly inserts.

Embodiment 11 utilizes recombination bacillus coli JM109 (pTrc99A-gshFap) synthesizing glutathion

With recombination bacillus coli JM109 (pTrc99A-gshFap) in the LB of the penbritin that contains 100mg/L substratum (containing peptone 10g, yeast extract 5g, NaCl 10g among the 1L) 37 ℃, the 220rpm shaking culture is to OD ₆₀₀Be 0.4-0.6, add 0.2mmol/L IPTG, in 30 ℃, the 220rpm shaking culture is induced 10h.Nutrient solution at the centrifugal 5min collecting cell of 8000rpm, is used 0.05mol/L, and the phosphoric acid buffer of pH7.0 washs centrifugal three times under same condition.Cell in-20 ℃ freezing 2 hours, carry out penetratingization processing.Take by weighing the 2g wet thallus and add in the 20mL reaction solution, reaction solution is 0.2mol/L (pH7.0) potassium phosphate buffer, contains Pidolidone 40mmol/L, Cys 20mmol/L, glycine 40mmol/L, MgCl ₂6H ₂O 20mmol/L, ATP 40mmol/L, in 37 ℃ of reactions 6 hours, the glutathione concentrations that obtains was 3.35g/L.

The GSH that e. coli jm109 (pTrc99a-gshFap) synthesizes than the reaction of wild-type e. coli JM109 similarity condition is high more than 40 times.The GSH measuring method that adopts is DTNB-circulation method (Tietze F (1969) Anal Biochem 27:502-522).Reaction solution is centrifugal, the concentration of GSH in the mensuration supernatant liquor; Reaction solution was heated 3 minutes in 100 ℃ of boiling water baths, and cool to room temperature is centrifugal rapidly, measures the GSH in the supernatant liquor, i.e. total GSH.Residual GSH seldom generally is no more than 20% of total GSH in the cell.

Embodiment 12 lactose-induced JM109 (pTrc99A-gshFap) synthesizing glutathions

The substratum that adopts contains: peptone 10g/L, yeast extract 5g/L, 50mM phosphoric acid buffer (pH 7.6), NH ₄Cl 50mM, Na ₂SO ₄5mM, MgSO ₄2mM, 0.2x metal ion (FeCl ₃50 μ mol/L, CaCl ₂20 μ mol/L, MnCl ₂4H ₂O 10 μ mol/L, ZnSO ₄7H ₂O 10 μ mol/L, CoCl ₂6H ₂O 2 μ mol/L, CuCl ₂2H ₂O 2 μ mol/L, NiCl ₂6H ₂O 2 μ mol/L, Na ₂MoO ₄5H ₂O 2 μ mol/L, Na ₂SeO ₃5H ₂O 2 μ mol/L, H ₃BO ₃2 μ mol/L), glycerine 54mM, glucose 2.8mM, alpha-lactose 5.6mM.

With e. coli jm109 (pTrc99A-gshFap) in this substratum in 30 ℃, 220rpm shaking culture 16 hours, centrifugation and the washing thalline, carry out penetrating processing 1 time by embodiment 1 freeze thawing.Take by weighing the 2g wet cell and add 20mL reaction solution composed as follows: Pidolidone 60mmol/L, Cys 40mmol/L, glycine 60mmol/L, MgCl ₂6H ₂O 20mmol/L, ATP 80mmol/L, Tris-HCl damping fluid 0.1mol/L (pH8.0), in 37 ℃ of reactions 6 hours, the glutathione concentrations that obtains was 7.1g/L.

Embodiment 13 provides the ATP synthesizing glutathion by glucose degradation

Adopt the cell culture processes of embodiment 12 and penetratingization of the cell treatment process of embodiment 1 to cultivate and process e. coli jm109 (pTrc99A-gshFap) cell, take by weighing the 2g wet cell and add in the 20mL reaction solution, reaction solution specifically consists of: Pidolidone 40m mol/L, Cys 20mmol/L, glycine 40mmol/L, MgCl ₂6H ₂O 20mmol/L, glucose 0.15mol/L, potassium phosphate buffer 0.2mol/L (pH8.0), in 37 ℃ of reactions 4 hours, the glutathione concentrations that obtains was 1.95g/L.

Embodiment 14 utilizes recombination bacillus coli BL21 (pET28a-gshFap) synthesizing glutathion

Press the method for embodiment 12 and cultivate e. coli bl21 (pET28a-gshFap), separate and washed cell, carry out penetrating processing.Taking by weighing the 2g wet cell adds in the 20mL reaction solution composed as follows: Pidolidone 40mmol/L, Cys 20mmol/L, glycine 40mmol/L, MgCl ₂6H ₂O 20mmol/L, glucose 0.15mol/L, potassium phosphate buffer 0.2mol/L (pH8.0) under 37 ℃, reacted 5 hours, and the glutathione concentrations that obtains is 2.23g/L.

SEQUENCE LISTING

＜110〉East China University of Science

＜120〉a kind of difunctional glutathione synthetase and utilize it to produce the method for gsh

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Lys Gly Asn Ile Val Thr Thr Ala His Pro Ser Val Phe Gly Asn Arg

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Ser Tyr His Pro Tyr Ile Gln Thr Asp Phe Ala Glu Ser Gln Leu Glu

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Leu Ile Thr Pro Pro Asn Asp Thr Leu Glu Asp Thr Tyr Arg Trp Leu

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Ser Ala Ile His Glu Val Thr Leu Arg Ser Leu Pro Asp Asp Glu Tyr

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Lys Glu Ala Gln Leu Asp Asn Glu Trp Asp Val Lys Tyr Arg Glu His

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Gln Thr Glu Tyr Arg Asp Lys Ile Ala Phe Arg Asn Ala Leu Tyr Met

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Glu Leu Ala Asn Asn Phe Leu Arg Tyr Gln Trp Ile Leu Val Tyr Leu

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Leu Ala Ala Thr Pro Thr Val Glu Ala Gln Tyr Phe Gly Lys Asn Ser

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Gly Tyr Val Asn Ala Pro His Ile Val Ile Asn His Asp Ser Leu Gln

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Gln Tyr Val Glu Ser Leu Glu His Phe Val Ala Thr Gly Asp Leu Leu

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Ala Arg Lys Leu Leu Glu Lys Gly Val Lys Tyr Ala Glu Phe Arg Leu

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Phe Asp Leu Asn Pro Phe Ser Pro Tyr Gly Ile Glu Leu Ala Asp Ala

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Lys Phe Ile His Leu Phe Leu Leu Ala Met Leu Trp Met Asp Glu Thr

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Ser Gly Gln Arg Glu Val Glu Ile Gly Thr Gln Lys Leu Tyr Gln Val

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Ala Leu Glu Asp Pro Arg Ser His Thr Ala Phe Gln Ala Glu Gly Glu

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Ala Ile Leu Asn Leu Met Leu Ala Met Leu Asp Asp Leu Ser Val Pro

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Gln Asn Glu Lys Asp Leu Leu Gln Gln Lys Leu Ala Gln Phe Ala Asp

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Pro Ser Gln Thr Val Asn Gly Arg Leu Leu Ala Ala Val Glu Gln Ala

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Gly Ser Tyr Lys Ala Leu Gly Ala Gln Leu Ala Gln Gln Tyr Lys Ala

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Gln Ala Phe Glu Arg Phe Tyr Ala Ile Ser Ala Phe Asp Asn Met Glu

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Gln Thr Ile His Glu Val Val Trp Arg Thr Leu Pro Glu Asp Glu Phe

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Ile Phe Pro Leu Ser Met Pro Ala Gly Leu Pro Pro Glu Lys Asp Ile

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Lys Val Ala Gln Leu Asp Asn Ser Val Asp Val Ala Tyr Arg Glu His

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Ala Glu Lys Glu Phe Tyr Ser Asn Val Arg Leu Arg Gly Ala Lys Lys

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Ala Arg Glu Phe Leu His Thr Gly Ile Gln Tyr Leu Glu Phe Arg Leu

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Phe Asp Leu Asn Pro Phe Glu Pro Tyr Gly Met Ser Leu His Asp Ala

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Gln Phe Ile His Tyr Phe Ile Leu Leu Met Ile Trp Leu Asp Glu Thr

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Ala Asp Gln Ala Gly Val Glu Leu Gly Lys Ala Arg Leu Leu Glu Val

325 330 335

Ala Phe Glu Asp Pro Arg Gln Glu Thr Val Tyr Arg Asp Glu Gly Glu

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Arg Ile Leu Asn Ala Leu Ile Thr Met Leu Lys Asp Leu Gly Thr Asp

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Glu Asn Ala Val Lys Ser Ala Gln Glu Lys Leu Ser Gln Phe Ala Tyr

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Pro Glu Arg Thr Leu Cys Ala Arg Leu Val Ala Ala Ile Glu Gln Ala

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Gly Gly Tyr Gln Lys Leu Gly Ala Glu Leu Ala Gln Arg Asn Lys Ala

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Gln Ala Phe Glu Arg Phe Tyr Ala Leu Thr Ala Phe Asp Asn Met Glu

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Leu Glu Gln Ala Val Ala Ser Tyr Ser Leu Phe Glu Gly Arg Ala Val

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Val Ile Lys Pro Lys Ser Thr Asn Tyr Gly Leu Gly Ile Thr Ile Phe

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Ala Ala Ile His Asp Val Ala Tyr Arg Ser Met Gly Asn Asn Glu Met

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Leu Asn Pro Phe Glu Thr Tyr Gly Ile Ser His Glu Gln Ala Glu Phe

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Leu Glu His Pro Leu Glu His Thr Gln Phe Lys Thr Asp Ala Glu Arg

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Ile Ile Asp Glu Met Glu His Leu Thr Gly Leu Leu Asp Ile Thr Val

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Ser Asn Thr Leu Phe Val Asn Leu Arg Glu Met Leu Thr Asp Pro Ser

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Lys Thr Leu Ala Gly Arg Leu Tyr Lys Glu Ile Ile Lys Ser Ser Gln

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Ser Gln Val Ala Ser Arg Ile Ala Lys Glu Asn Tyr Lys Lys Ala Trp

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Asp Lys Pro Tyr Gln Leu Ser Gly Phe Thr Asp Met Glu Leu Ser Thr

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Gln Ile Leu Met Phe Asp Ala Ile Gln Gln Gly Leu Gln Val Asp Val

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Leu Asp Arg Gln Asp Gln Phe Leu Lys Leu Gln Leu Gly Asn His Val

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Glu Tyr Val Lys Asn Gly Asn Met Thr Ser Lys Asp Ser Tyr Ile Ser

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Pro Leu Ile Met Glu Asn Lys Thr Val Thr Lys Lys Ile Leu Gln Gln

485 490 495

His Gly Phe Arg Val Pro Ile Gly Glu Glu Phe Ser Asp Ile Glu Lys

500 505 510

Ala Leu Arg Ser Tyr Asp Ile Phe Ala Gly Lys Pro Phe Val Val Lys

515 520 525

Pro Lys Thr Thr Asn Tyr Gly Leu Gly Ile Ser Ile Phe Lys Glu Asn

530 535 540

Gly Ala Ser Tyr Glu Asp Tyr Gln Lys Ala Leu Thr Ile Ala Phe Lys

545 550 555 560

Glu Asp Ser Ser Val Leu Ile Glu Glu Phe Ile Asn Gly Thr Glu Tyr

565 570 575

Arg Phe Phe Val Leu Asp Gly Lys Val Ser Ala Val Leu Leu Arg Ile

580 585 590

Pro Ala Asn Val Ile Gly Asp Gly Ser His Thr Ile Glu Glu Leu Val

595 600 605

Ala Gln Lys Asn Leu Asn Ser Leu Arg Gly Met Asp His Arg Thr Pro

610 615 620

Leu Glu Asn Ile Gln Leu Gly Glu Leu Glu Val Leu Met Leu Lys Ala

625 630 635 640

Gln Gly Tyr Arg Lys Asp Ser Ile Pro Thr Ser Asp Glu Ile Val Phe

645 650 655

Leu Arg Glu Asn Ser Asn Val Ser Thr Gly Gly Asp Ser Ile Asp Met

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Thr Asp Gln Ile Pro Asp Asp Tyr Lys Lys Ile Ala Val Asp Ala Val

675 680 685

Ser Ala Leu Gly Ala Asn Ile Ser Gly Ile Asp Leu Ile Ile Glu Asn

690 695 700

Thr Glu Val Pro Ala Ala Asn Lys Asn Ala Tyr Gly Ile Ile Glu Ala

705 710 715 720

Asn Phe Asn Pro Ser Met Tyr Met His Ile Tyr Pro Tyr Lys Gly Lys

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Ser Arg Arg Leu Thr Ile Cys Ile Leu His Tyr Leu Phe Pro Glu Leu

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Pro Lys Lys

755

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Met Met Thr Ile Asn Gln Leu Leu Gln Lys Leu Asp Thr Ala Ser Pro

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Ile Leu Gln Ala Thr Phe Gly Leu Glu Arg Glu Asn Leu Arg Val Thr

20 25 30

Thr Asp Gly His Leu Ala Gln Thr Ala His Pro Ser Gln Leu Gly Ser

35 40 45

Arg Asn Phe His Pro Thr Ile Gln Thr Asp Phe Ser Glu Gln Gln Leu

50 55 60

Glu Leu Ile Thr Pro Ile Ala His Ser Thr Lys Glu Ala Arg Arg Leu

65 70 75 80

Leu Gly Ala Ile Ser Asp Val Ala Gly Arg Ser Ile Asp Gln Asn Glu

85 90 95

Arg Leu Trp Pro Met Ser Met Pro Pro Gln Leu Thr Glu Glu Glu Ile

100 105 110

Ala Ile Ala His Leu Glu Asn Asp Tyr Glu Arg His Tyr Arg Glu Gly

115 120 125

Leu Ala Lys Lys Tyr Gly Lys Lys Leu Gln Ala Ile Ser Gly Ile His

130 135 140

Tyr Asn Met Glu Leu Gly Lys Asp Leu Val Thr Ser Leu Phe Gln Val

145 150 155 160

Ser Ser Tyr His Ser Leu Lys Asp Phe Lys Asn Asp Leu Tyr Leu Lys

165 170 175

Leu Ala Arg Asn Phe Leu Arg Phe Arg Trp Ile Leu Thr Tyr Leu Tyr

180 185 190

Gly Ala Ala Pro Trp Ala Glu Ala Gly Phe Tyr Ser Gln Glu Ile Ser

195 200 205

Gln Pro Ile Arg Ser Phe Arg Asn Ser Asp Tyr Gly Tyr Val Asn Asp

210 215 220

Glu Asn Ile Gln Val Ser Tyr Ala Ser Leu Glu Gln Tyr Ile Thr Asp

225 230 235 240

Ile Glu Asn Tyr Val Gln Ser Gly Glu Leu Ser Ala Glu Lys Glu Phe

245 250 255

Tyr Ser Ala Val Arg Phe Arg Gly Gln Lys His Asn His Ala Tyr Leu

260 265 270

Glu Gln Gly Ile Thr Tyr Leu Glu Phe Arg Cys Phe Asp Leu Asn Pro

275 280 285

Phe Asp His Leu Gly Ile Ser Gln Glu Thr Leu Asp Thr Val His Leu

290 295 300

Phe Leu Leu Ser Leu Leu Trp Leu Asp Asp Val Glu Asn Val Asp Thr

305 310 315 320

Ala Leu Lys Ala Ala His Asp Leu Asn Gln Lys Ile Ala Cys Ser His

325 330 335

Pro Leu Thr Ala Leu Pro Asp Glu Ala Asp Ser Ser Ala Leu Leu Gln

340 345 350

Ala Met Glu Glu Leu Ile Gln His Phe Glu Leu Pro Thr Tyr Tyr Gln

355 360 365

Thr Leu Leu Gln Gln Leu Lys Glu Ala Leu Leu Asn Pro Gln Leu Thr

370 375 380

Leu Ser Gly Gln Leu Leu Pro His Ile Gln Gln Asp Ser Leu Met Ala

385 390 395 400

Phe Gly Leu Glu Lys Ala Glu Glu Tyr His Arg Tyr Ala Trp Thr Ala

405 410 415

Pro Tyr Ala Leu Lys Gly Tyr Glu Asn Met Glu Leu Ser Thr Gln Met

420 425 430

Leu Leu Phe Asp Ala Ile Gln Lys Gly Leu Asn Val Asp Ile Leu Asp

435 440 445

Glu Asn Asp Gln Phe Leu Lys Leu Trp His Gly His His Val Glu Tyr

450 455 460

Val Lys Asn Gly Asn Met Thr Ser Lys Asp Asn Tyr Val Ile Pro Leu

465 470 475 480

Ala Met Ala Asn Lys Thr Val Thr Lys Lys Ile Leu Ala Glu Ala Asp

485 490 495

Phe Pro Val Pro Ala Gly Ala Glu Phe Ser Ser Leu Glu Glu Gly Leu

500 505 510

Ala Tyr Tyr Pro Leu Ile Arg Asp Arg Gln Ile Val Val Lys Pro Lys

515 520 525

Ser Thr Asn Phe Gly Leu Gly Ile Ser Ile Phe Gln Glu Pro Ala Ser

530 535 540

Leu Glu Ser Tyr Arg Lys Ala Leu Glu Ile Ala Phe Ser Glu Asp Ala

545 550 555 560

Ala Val Leu Val Glu Glu Phe Ile Ala Gly Thr Glu Tyr Arg Phe Phe

565 570 575

Val Leu Asp Gly Gln Cys Glu Ala Val Leu Leu Arg Val Ala Ala Asn

580 585 590

Val Val Gly Asp Gly Gln His Thr Val Arg Glu Leu Val Ala Ile Lys

595 600 605

Asn Asp Asn Pro Leu Arg Gly Arg Asp His Arg Ser Pro Leu Glu Ile

610 615 620

Ile Glu Leu Gly Asp Ile Glu Leu Leu Met Leu Asp Gln Gln Gly Tyr

625 630 635 640

Gly Pro Asp Asp Ile Leu Pro Asp Gly Val Lys Val Asp Leu Arg Arg

645 650 655

Asn Ser Asn Ile Ser Thr Gly Gly Asp Ser Ile Asp Val Thr Asp Ser

660 665 670

Met His Pro Ser Tyr Lys Glu Leu Ala Ala Asp Met Ala Lys Ala Met

675 680 685

Gly Ala Trp Ala Cys Gly Val Asp Leu Ile Ile Pro Asp Ser Ser Ala

690 695 700

Ile Ser Thr Lys Glu Asn Pro Asn Cys Thr Cys Ile Glu Leu Asn Phe

705 710 715 720

Asn Pro Ser Met Tyr Met His Thr Tyr Cys Ala Glu Gly Pro Gly Gln

725 730 735

Ser Ile Thr Pro Lys Ile Leu Ala Lys Leu Phe Pro Glu Met Asp

740 745 750

<210>5

<211>751

<212>PRT

<213>Streptococcus gordonii

<400>5

Met Thr Ile Asn Gln Leu Leu Gln Lys Leu Asp Thr Ser Ser Pro Ile

1 5 10 15

Leu Gln Ala Thr Phe Gly Leu Glu Arg Glu Asn Leu Arg Val Thr Pro

20 25 30

Asp Gly His Leu Ala Gln Thr Ala His Pro Asn Lys Leu Gly Ser Arg

35 40 45

Asn Phe His Pro Ser Ile Gln Thr Asp Phe Ser Glu Gln Gln Leu Glu

50 55 60

Leu Ile Thr Pro Val Ala His Ser Thr Lys Glu Ala Arg Arg Leu Leu

65 70 75 80

Gly AlaIle Ser Asp Val Ala Ala Arg Ser Ile Asp Gln Asn Glu Arg

85 90 95

Leu Trp Pro Leu Ser Met Pro Pro Gln Leu Thr Glu Glu Glu Ile Val

100 105 110

Ile Ala Arg Leu Glu Asn Ala Tyr Glu Arg His Tyr Arg Glu Gly Leu

115 120 125

Ala Glu Lys Tyr Gly Lys Lys Leu Gln Ala Ile Ser Gly Ile His Tyr

130 135 140

Asn Met Glu Leu Gly Lys Asp Leu Val Thr Ala Leu Phe Arg Val Ser

145 150 155 160

Ser His His Ser Leu Lys Asp Phe Lys Asn Asp Leu Tyr Leu Lys Leu

165 170 175

Ala Arg Asn Phe Leu Arg Phe Arg Trp Ile Leu Thr Tyr Leu Tyr Gly

180 185 190

Ala Ala Pro Leu Ala Glu Ala Gly Phe Tyr Ser Gln Asp Ser Ser Gln

195 200 205

Pro Ile Arg Ser Phe Arg Asn Ser Asp Tyr Gly Tyr Val Asn Asp Glu

210 215 220

Asn Ile Lys Val Ser Tyr Ala Ser Leu Glu Gln Tyr Val Thr Asp Ile

225 230 235 240

Glu Asn Tyr Val Gln Ser Gly Lys Leu Ser Ala Glu Lys Glu Phe Tyr

245 250 255

Ser Ala Val Arg Phe Arg Gly Gln Lys His Asn Arg Val Tyr Leu Glu

260 265 270

Gln Gly Ile Thr Tyr Leu Glu Phe Arg Cys Phe Asp Leu Asn Pro Phe

275 280 285

Asp His Leu Gly Ile Ser Gln Glu Thr Leu Asp Thr Val His Leu Phe

290 295 300

Leu Leu Ser Leu Leu Trp Leu Asp Asp Val Glu Asn Val Asp Val Ala

305 310 315 320

Leu Lys Ala Ala His Asp Leu Asn Gln Lys Ile Ala Cys Ser His Pro

325 330 335

Leu Thr Ala Leu Pro Asp Glu Ala Asp Ser Ser Thr Leu Leu Gln Ala

340 345 350

Met Glu Glu Leu Ile Gln His Phe Glu Leu Pro Thr Tyr Tyr Gln Ser

355 360 365

Leu Leu Glu Gln Leu Lys Glu Ser Leu Leu Asn Pro Gln Leu Thr Leu

370 375 380

Ser Gly Gln Leu Leu Pro His Ile Gln Gln Asp Ser Leu Met Asp Phe

385 390 395 400

Gly Leu Glu Lys Ala Glu Glu Tyr His His Tyr Ala Trp Ser Ala Pro

405 410 415

Tyr Ala Leu Lys Gly Tyr Glu Asn Met Glu Leu Ser Thr Gln Met Leu

420 425 430

Leu Phe Asp Ala Ile Gln Arg Gly Leu Asn Phe Glu Ile Leu Asp Glu

435 440 445

Asn Asp Gln Phe Leu Lys Leu Trp Tyr Gly Pro His Val Glu Tyr Val

450 455 460

Lys Asn Gly Asn Met Thr Ser Lys Asp Asn Tyr Val Ile Pro Leu Ala

465 470 475 480

Met Ala Asn Lys Ile Val Thr Lys Lys Ile Leu Ala Ala Ala Asp Phe

485 490 495

Pro Val Pro Ala Gly Ala Glu Phe Thr Thr Leu Glu Glu Gly Ile Leu

500 505 510

Tyr Tyr Pro Leu Ile Lys Asp Arg Gln Ile Val Val Lys Pro Lys Ser

515 520 525

Thr Asn Phe Gly Leu Gly Ile Ser Ile Phe Gln Glu Pro Ala Ser Leu

530 535 540

Glu Ala Tyr Arg Lys Ala Leu Glu Ile Ala Phe Ser Glu Asp Thr Ala

545 550 555 560

Val Leu Val Glu Glu Phe Ile Ala Gly Thr Glu Tyr Arg Phe Phe Val

565 570 575

Leu Asp Gly Gln Cys Glu Ala Val Leu Leu Arg Val Ala Ala Asn Val

580 585 590

Val Gly Asp Gly Gln His Thr Val Arg Glu Leu Val Ala Ile Lys Asn

595 600 605

Asp Asn Pro Leu Arg Gly Arg Asp His Arg Ser Pro Leu Glu Ile Ile

610 615 620

Glu Leu Gly Asp Ile Glu Leu Leu Met Leu Asp Gln Gln Gly Tyr Gly

625 630 635 640

Pro Asp Asp Ile Leu Pro Ala Gly Val Lys Val Asp Leu Arg Arg Asn

645 650 655

Ser Asn Ile Ser Thr Gly Gly Asp Ser Ile Asp Val Thr Asp Ser Met

660 665 670

His Pro Ser Tyr Lys Glu Leu Ala Ala Asp Met Ala Lys Ala Met Gly

675 680 685

Ala Trp Ala Cys Gly Val Asp Leu Ile Ile Pro Asp Ser Ser Ala Ile

690 695 700

Ser Thr Lys Glu Asn Pro Asn Cys Thr Cys Ile Glu Leu Asn Phe Asn

705 710 715 720

Pro Ser Met Tyr Met His Thr Tyr Cys Ala Glu Gly Pro Gly Gln Ser

725 730 735

Ile Thr Pro Lys Ile Leu Ala Lys Leu Phe Pro Glu Met Asp Leu

740 745 750

<210>6

<211>753

<212>PRT

<213>Streptococcus uberis

<400>6

Met Leu Asn Gln Leu Leu Gln Lys Leu Pro Gln Asn Thr Asp Ile Leu

1 5 10 15

Glu Ala Thr Phe Gly Leu Glu Arg Glu Gly Leu Arg Leu Thr Gln Glu

20 25 30

Gly Thr Leu Ala Gln Thr Asp His Pro Lys Ala Leu Gly Ser Arg Ser

35 40 45

Phe His Pro Tyr Ile Gln Thr Asp Phe Ser Glu Gln Gln Leu Glu Leu

50 55 60

Ile Thr Pro Ile Ser Gln Ser Thr Gln Glu Ala Arg Arg Arg Leu Gly

65 70 75 80

Ala Ile Phe Asp Val Ala Gln Arg Ser Leu Glu Glu Asp Gln Val Ile

85 90 95

Trp Pro Leu Ser Ile Pro Pro Tyr Ile Glu Glu Asp Gln Ile Gln Ile

100 105 110

Ala Lys Leu Asp Lys Ala Glu Glu Val Arg Tyr Arg Glu Gln Leu Ala

115 120 125

Gln Thr Tyr Gly Lys Lys Leu Gln Ser Ile Ser Gly Ile His Tyr Asn

130 135 140

Ile Glu Leu Gly Lys Asn Leu Thr Gln Asn Leu Phe Ala Leu Ser Asp

145 150 155 160

Gln Glu Asp Leu Lys Asp Phe Lys Asn Ala Ile Tyr Met Lys Leu Ala

165 170 175

Arg Gln Phe Leu Asn Tyr Gln Trp Leu Val Thr Tyr Leu Phe Gly Ala

180 185 190

Ser Pro Leu Ala Glu Lys Asp Phe Tyr Pro Gln Met Pro Thr Glu Leu

195 200 205

Val Arg Ser Leu Arg Ala Ser Arg Arg Tyr Gly Tyr Ser Asn His Glu

210 215 220

Glu Leu Ser Ile Ser Phe Ser Ser Leu Glu Asn Tyr Val Lys Asp Met

225 230 235 240

Glu Asn Ala Leu Ala Thr Gly Ile Leu Ser Leu Glu Lys Glu Phe Tyr

245 250 255

Ser Pro Val Arg Leu Arg Gly Ser Lys His Ser Arg Asp Tyr Leu Ser

260 265 270

Glu Gly Ile Thr Tyr Leu Glu Phe Arg Asn Phe Asp Ile Asn Pro Phe

275 280 285

Asp Lys Leu Gly Ile Ser Gln Lys Thr Leu Asp Ser Phe His Leu Phe

290 295 300

Leu Leu Ser Leu Leu Trp Leu Asp Asp Leu Lys Asp Ser Asp Gln Glu

305 310 315 320

Leu Thr Lys Ala Arg Gln Ile Asn Glu Glu Val Ala Leu Ala His Pro

325 330 335

Met Ser Pro Leu Pro Asp Pro Glu Leu Ala Arg Pro Val Leu Lys Ala

340 345 350

Met Thr Glu Leu Ile Gln His Phe Gly Leu Asp Ala Tyr Tyr Gln Asp

355 360 365

Leu Val Ala Asp Leu Glu Leu Ala Ile Lys Glu Pro Glu Arg Thr Ile

370 375 380

Ser Gly His Leu Phe Gly Lys Ile Ala Asn Ala Ser Leu Ala Ala Phe

385 390 395 400

Gly Leu Asp Gln Ala Lys Thr Tyr His Gln Glu Ala Thr Gln Ala Pro

405 410 415

Tyr Ala Leu Lys Gly Tyr Glu Ser Met Glu Leu Ser Thr Gln Met Leu

420 425 430

Met Phe Asp Ala Ile Gln Lys Gly Leu His Leu Asp Ile Leu Asp Glu

435 440 445

Asn Asp Gln Phe Leu Lys Ile Trp His Gly Asp His Val Glu Tyr Val

450 455 460

Lys Asn Gly Asn Met Thr Ser Arg Asp Asn Tyr Val Val Pro Leu Ala

465 470 475 480

Met Ala Asn Lys Thr Val Thr Lys Lys Ile Leu Asp Gln Ala Gly Phe

485 490 495

Pro Thr Pro Lys Gly Gln Glu Phe Ala Ser Lys Asp Gln Ala Ile Arg

500 505 510

Tyr Phe Asn Gln Ile Ala Asp Lys Ala Ile Val Val Lys Pro Lys Ser

515 520 525

Thr Asn Phe Gly Leu Gly Ile Ser Ile Phe Gln Lys Pro Ala Ser Gln

530 535 540

Ala Asp Tyr Glu Lys Ala Val Glu Ile Ala Phe Ala Glu Asp Arg Asp

545 550 555 560

Ile Leu Val Glu Glu Phe Ile Ala Gly Thr Glu Tyr Arg Phe Phe Thr

565 570 575

Leu Asn Gly Lys Cys Glu Ala Val Leu Leu Arg Leu Pro Ala Asn Val

580 585 590

Val Gly Asp Gly Ile His Thr Val Lys Glu Leu Ile Asp Leu Lys Asn

595 600 605

Gln Asn Pro Leu Arg Gly Leu Asp His Arg Ser Pro Leu Glu Lys Ile

610 615 620

Gln Leu Gly Asp Ile Glu Lys Leu Met Leu Ala Gln Glu Gly Tyr Gly

625 630 635 640

Pro Asp Ser Val Leu Ala Lys Gly Val Lys Val Glu Leu Arg Arg Asn

645 650 655

Ser Asn Ile Ser Thr Gly Gly Asp Ser Val Asp Met Thr Asp Glu Met

660 665 670

Asp Pro Ser Tyr Lys Ala Leu Ala Ala Gln Met Ala Asp Ala Met Gly

675 680 685

Ala Trp Val Cys Gly Val Asp Leu Ile Ile Pro Asp Pro Ser Gln Lys

690 695 700

Ala Ser Leu Glu Glu Pro Asn Cys His Cys Ile Glu Leu Asn Phe Asn

705 710 715 720

Pro Ser Met Tyr Met His Thr Tyr Cys Gln Glu Gly Pro Gly Gln Ala

725 730 735

Ile Thr Pro Lys Ile Leu Gln Ala Leu Phe Pro Glu Leu Pro Ile Tyr

740 745 750

Pro

<210>7

<211>754

<212>PRT

<213>Streptococcus thermophilus

<400>7

Met Thr Leu Asn Gln Leu Leu Gln Lys Leu Glu Ala Thr Ser Pro Ile

1 5 10 15

Leu Gln Ala Asn Phe Gly Ile Glu Arg Glu Ser Leu Arg Val Asp Arg

20 25 30

Gln Gly Gln Leu Val His Thr Pro His Pro Ser Cys Leu Gly Ala Arg

35 40 45

Ser Phe His Pro Tyr Ile Gln Thr Asp Phe Cys Glu Phe Gln Met Glu

50 55 60

Leu Ile Thr Pro Val Ala Lys Ser Thr Thr Glu Ala Arg Arg Phe Leu

65 70 75 80

Gly Ala Ile Thr Asp Val Ala Gly Arg Ser Ile Ala Thr Asp Glu Val

85 90 95

Leu Trp Pro Leu Ser Met Pro Pro Arg Leu Lys Ala Glu Glu Ile Gln

100 105 110

Val Ala Gln Leu Glu Asn Asp Phe Glu Arg His Tyr Arg Asn Tyr Leu

115 120 125

Ala Glu Lys Tyr Gly Thr Lys Leu Gln Ala Ile Ser Gly Ile His Tyr

130 135 140

Asn Met Glu Leu Gly Lys Asp Leu Val Glu Ala Leu Phe Gln Glu Ser

145 150 155 160

Gly Gln Thr Asp Met Ile Ala Phe Lys Asn Ala Leu Tyr Leu Lys Leu

165 170 175

Ala Gln Asn Tyr Leu Arg Tyr Arg Trp Val Ile Thr Tyr Leu Phe Gly

180 185 190

Ala Ser Pro Ile Ala Glu Gln Gly Phe Phe Asp Gln Glu Val Pro Glu

195 200 205

Pro Val Arg Ser Phe Arg Asn Ser Asp His Gly Tyr Val Asn Lys Glu

210 215 220

Glu Ile Gln Val Ser Phe Val Ser Leu Glu Asp Tyr Val Ser Ala Ile

225 230 235 240

Glu Thr Tyr Ile Glu Gln Gly Asp Leu Asn Ala Glu Lys Glu Phe Tyr

245 250 255

Ser Ala Val Arg Phe Arg Gly Gln Lys Val Asn Arg Ser Phe Leu Asp

260 265 270

Lys Gly Ile Thr Tyr Leu Glu Phe Arg Asn Phe Asp Leu Asn Pro Phe

275 280 285

Glu Arg Ile Gly Ile Ser Gln Thr Thr Met Asp Thr Val His Leu Leu

290 295 300

Ile Leu Ala Phe Leu Trp Leu Asp Ser Pro Glu Asn Val Asp Gln Ala

305 310 315 320

Leu Ala Gln Gly His Ala Leu Asn Glu Lys Ile Ala Leu Ser His Pro

325 330 335

Leu Glu Pro Leu Pro Ser Glu Ala Lys Thr Gln Asp Ile Val Thr Ala

340 345 350

Leu Asp Gln Leu Val Gln His Phe Gly Leu Gly Asp Tyr His Gln Asp

355 360 365

Leu Val Lys Gln Val Lys Ala Ala Phe Ala Asp Pro Asn Gln Thr Leu

370 375 380

Ser Ala Gln Leu Leu Pro Tyr Ile Lys Asp Lys Ser Leu Ala Glu Phe

385 390 395 400

Ala Leu Asn Lys Ala Leu Ala Tyr His Asp Tyr Asp Trp Thr Ala His

405 410 415

Tyr Ala Leu Lys Gly Tyr Glu Glu Met Glu Leu Ser Thr Gln Met Leu

420 425 430

Leu Phe Asp Ala Ile Gln Lys Gly Ile His Phe Glu Ile Leu Asp Glu

435 440 445

Gln Asp Gln Phe Leu Lys Leu Trp His Gln Asp His Val Glu Tyr Val

450 455 460

Lys Asn Gly Asn Met Thr Ser Lys Asp Asn Tyr Val Val Pro Leu Ala

465 470 475 480

Met Ala Asn Lys Thr Val Thr Lys Lys Ile Leu Ala Asp Ala Gly Phe

485 490 495

Pro Val Pro Ser Gly Asp Glu Phe Thr Ser Leu Glu Glu Gly Leu Ala

500 505 510

Tyr Tyr Pro Leu Ile Lys Asp Lys Gln Ile Val Val Lys Pro Lys Ser

515 520 525

Thr Asn Phe Gly Leu Gly Ile Ser Ile Phe Gln Glu Pro Ala Ser Leu

530 535 540

Asp Asn Tyr Gln Lys Ala Leu Glu Ile Ala Phe Ala Glu Asp Thr Ser

545 550 555 560

Val Leu Val Glu Glu Phe Ile Pro Gly Thr Glu Tyr Arg Phe Phe Ile

565 570 575

Leu Asp Gly Arg Cys Glu Ala Val Leu Leu Arg Val Ala Ala Asn Val

580 585 590

Ile Gly Asp Gly Lys His Thr Ile Arg Glu Leu Val Ala Gln Lys Asn

595 600 605

Ala Asn Pro Leu Arg Gly Arg Asp His Arg Ser Pro Leu Glu Ile Ile

610 615 620

Glu Leu Gly Asp Ile Glu Gln Leu Met Leu Ala Gln Gln Gly Tyr Thr

625 630 635 640

Pro Asp Asp Ile Leu Pro Glu Gly Lys Lys Val Asn Leu Arg Arg Asn

645 650 655

Ser Asn Ile Ser Thr Gly Gly Asp Ser Ile Asp Ile Thr Glu Thr Met

660 665 670

Asp Ser Ser Tyr Gln Glu Leu Ala Ala Ala Met Ala Thr Ser Met Gly

675 680 685

Ala Trp Ala Cys Gly Val Asp Leu Ile Ile Pro Asp Glu Thr Gln Ile

690 695 700

Ala Thr Lys Glu Asn Pro His Cys Thr Cys Ile Glu Leu Asn Phe Asn

705 710 715 720

Pro Ser Met Tyr Met His Thr Tyr Cys Ala Glu Gly Pro Gly Gln Ala

725 730 735

Ile Thr Thr Lys Ile Leu Asp Lys Leu Phe Pro Glu Ile Val Ala Gly

740 745 750

Gln Thr

<210>8

<211>2274

<212>DNA

<213>Actinobacillus pleuropneumonia

<400>8

atgaaattac aacaactgat taaaactcat caccttggtt tactatttca acaaggtaaa 60

tttggcatcg aaaaagaaag ccaacgcatt gataataaag ggaatattgt tactaccgcc 120

catcctagcg tttttggtaa ccgcagttat catccgtata ttcaaaccga ttttgcagaa 180

agtcagttag aacttatcac accaccaaac gatacattgg aagacacata tcgttggcta 240

tcggctattc acgaggtaac gttacgttcg ttgcccgatg atgaatatat tttcccattc 300

agcatgcctg ccggtttacc gccggaattc gaaatcaaag aagcacaatt agataacgaa 360

tgggacgtga aatatcgtga acacctttct gccatttatg gcaaatacaa gcaaatggtg 420

agcggtattc actataattt ccaaatttcc gaagaatttg tcgaaagcac atttgcatta 480

caaacggaat accgcgataa aattgcgttc cgcaatgcgc tatatatgga attagccaat 540

aactttttac gttatcaatg gattttagtt tatttgcttg ccgcaacccc aactgtagaa 600

gcgcaatatt tcggtaaaaa ctcaccgctt gcagaagggc aattagtacg tagtttacgt 660

tccggccctt atggttatgt aaatgcgcca catattgtga tcaaccacga cagcttgcaa 720

caatatgtcg agtcgctaga acattttgta gcaaccggcg atttgttggc agaaaaagaa 780

ttctattcaa acgttcgttt acgtggtgcg aaaaaagcac gcaaattgct tgagaaaggg 840

gttaaatatg cggaattccg tttatttgat cttaatccgt tctcgcctta cggtatcgag 900

cttgcagacg cgaaatttat tcatctgttc ttacttgcga tgttgtggat ggatgaaaca 960

agcggtcaaa gagaagtcga aatcggcaca caaaaactat accaagttgc ccttgaagat 1020

cctcgttcgc acactgcgtt ccaagcagag ggtgaggcga tccttaacct gatgctggca 1080

atgctggacg atctttctgt accacaaaac gagaaagatt tattacaaca aaaactggca 1140

caatttgccg atcctagtca aacggtaaac ggtcgtttat tagccgcagt cgaacaagcc 1200

ggcagctata aagctctcgg tgcacaactt gctcaacaat ataaagcgca agcattcgag 1260

cgtttttatg cgatttccgc tttcgataat atggagcttt ctacacaggc tttgttattt 1320

gatgcgatcc aacaaggctt acagatcgaa ttgcttgatg aaaacgatca gttcctcgca 1380

ctcaaattcg gcgatcatct cgaatatgtg aaaaacggca atatgaccag ccacgatcag 1440

tatatttcgc cattaattat ggaaaacaaa gtcgtaacca aaaaagtgtt ggcgaaagcc 1500

ggttttaatg tgccgaaaag tgttgaattt acctctgtag aacaagcggt ggcacactat 1560

ccgttatttg aaggtaaagc ggtggtaatt aagccgaaat caactaatta cggcttaggt 1620

attacaattt tccagcaagg cgtgacggat aaagccgact ttgccaaagc gattgaaatt 1680

gcgttccgtg aagataaaga agtgatggtg gaagactatt tagtcggcac cgaataccgt 1740

ttctttgtgt taggcgatga aacactggcg gtattgttac gtgtgccggc aaatgtgaaa 1800

ggtgactgca tacatacagt gcgtgaattg gtggaagcga aaaacagtga tccgctacga 1860

ggtgacggct ctcgttcacc attgaagaaa atcgccctcg gtgacattga attgcttcag 1920

cttaaagagc aaggtttaac gcctgattcg attccggctg acgggcaaat cgtacaatta 1980

cgtgccaact ctaatattag taccggcggc gattcaatcg atatgaccga tcaaatgcat 2040

gacagttata aacaattagc ggtcggtatt gccaaagaga tgggggcaaa agtctgcggt 2100

gtggatttaa tcattccgga tttaaccaaa gccgccgagc cgtctctgcg ttcatggggt 2160

gtgattgaag caaactttaa tccgatgatg atgatgcata ttttccctta ccaaggaaaa 2220

tctcgccgct taaccaaagc cgtgttaaaa atgctgttcc cagaactgcc ttaa 2274

<210>9

<211>2280

<212>DNA

<213>Actinobacillus succinogenes

<400>9

atgaatttac ggcatattat taaacaaaat catcttgaat tgttattcca acaggggtct 60

ttcgggctgg aaaaagagag ccagcgcgtc cgtcacgacg gttcggtggt gacatccgcc 120

cacccgaagg ctttcggtaa ccgctctttt catccgtata tacaaacgga ttttgccgaa 180

agccagcttg aattgattac accgccgaat aagaaactgg aagatacgtt tcgttggctg 240

caaactatcc atgaagtggt atggcgaacc ttgccggaag acgaattcat ctttcccctg 300

agcatgcctg cgggtttgcc gccggaaaaa gacattaaag tcgcccaact ggataattct 360

gttgatgtgg cataccgcga acatttagtg aaatcttacg gtgcctacaa gcaaatggta 420

agcggaattc attataattt tcagcttgat cccgcgttga ttgaggcctt attcaaggca 480

cagagcgatt gtcgaagtgc ggtggaattt cagaacagtt tatatctgaa aatggcaaaa 540

aatttcctgc gctatcaatg gattctgttg tatttgttat ccgctactcc gacggtggac 600

gccaattact tcagagaggg aacagcgctg aaaccgaatc aatatgtgcg cagcctgcgt 660

tccagtcaat acgggtatgt caatgcgcct gaaatcgtgg tgtctttcga cagcattccc 720

caatatgtgg aaacccttga gcactgggtg aattcgggca aattgattgc ggaaaaagaa 780

ttttattcca atgtacgttt gcgcggggcg aaaaaagccc gcgaattttt gcatacgggg 840

atccaatatc tggaattccg tttattcgat ttaaatccgt tcgaaccgta cggcatgagc 900

ttacatgatg cgcaatttat ccactatttt atcctgttga tgatttggct ggatgaaacg 960

gcggatcaag ccggcgtaga attaggaaaa gcgcgtttgc tggaagtcgc gtttgaagac 1020

ccgcgtcagg aaaccgttta tcgcgacgaa ggcgaacgga ttttgaatgc tttaattacc 1080

atgttaaaag acctcggcac agatgaaaat gcggtgaaaa gcgcacagga aaaactgagc 1140

cagtttgctt atccggaacg aacgctgtgc gcccggttgg ttgccgccat cgaacaagcc 1200

ggcggctatc aaaaactggg ggcggaattg gcgcagcgta ataaagcgca ggctttcgaa 1260

cgtttttacg ccctgaccgc tttcgataat atggaattgt ctacgcaggc tttaatgttt 1320

gatgcgattc aaaaagggtt acaaatggaa attctggatg aaaacgatca gtttctgcgt 1380

ctgcaattcg gcgagcattt cgaatacgtg aaaaacggca acatgacaag ccgcgacagc 1440

tacatttcgc cgcttattat ggaaaataaa gtggtgacta aaaaagtatt ggataaagcc 1500

ggattcaacg taccgaaaag tctggaattc actacccttg aacaagcggt ggcaagttac 1560

agcctgtttg aagggcgcgc tgtggtgatt aaaccgaaat ccaccaatta cggcttaggc 1620

atcaccattt ttcaacaggg cgtgcatgat cgcgacgatt tcgccaaagc cgttgaaatc 1680

gcgttccgtg aagataaaga agtgatggtg gaagattatt tgagcggtac ggaataccgg 1740

tttttcgtgt tgggcgatga aacccttgcg gtattgctgc gcgtgccggc gaatgtcgtg 1800

ggtgacggtg tacgcacggt ggcggaactg gttgcggcga aaaacgatca tccgttacgt 1860

ggcgacggca gccgtactcc gctgaaaaaa atcacgttag gcgagattga acggttgcag 1920

cttaaagagc aaggtttgac cgtcgacagc gtgccggccg aagggctgct tgtacaactt 1980

cgggcaaact ctaatatcag caccggcggc gatagtatcg acatgacgga tgaaatgcat 2040

ccgagctata aagcgcttgc cgtagacatt accaaagcca tgggggcggc ggtttgcggt 2100

gtggatctta ttattccgga tttgaataag ccggcggaaa accgcttatc gtcttgggga 2160

gttatcgaag ctaatttcaa tccgatgatg atgatgcaca ttttcccata tcaaggaaaa 2220

tccagacgtt tgacgcagaa tgtgattaag atgctgtttc cggaattatt aaatgcatga 2280

<210>10

<211>2268

<212>DNA

<213>Bacillus cereus

<400>10

atggaaatga aaaaaatgtt aaacaatgac cgtattaaac catatttatt aaaagcacgt 60

tttggagttg aaaaagaaag tcaacgagtt gacctatcag gaagtttagc taaaacggaa 120

catcccaaaa gtatttcagt aagagatgaa catccttata ttcagcgaga tttctctgaa 180

acacaaatgg aattaatcac accagttact gagacactag gagatttatt taattattta 240

gcagctatcc atgatgttgc ctatcgttct atgggaaata acgaaatgct ttggccatta 300

agcatgccac cacagttgcc cgaaaaagaa gaagatattg ttattgcgaa acttaataat 360

catgaaaatg ttttatatcg tcgatattta tctaattctt atggtcgacg aaaacaaatg 420

attagtggta ttcattacaa ttttgaattt agcgataatt tgattcaggc attatttgaa 480

ttacaatcag aaataaaaga ttatcatcaa tttaaaacag aaatttatct gaaagtcaca 540

aggaattatt tacactatcg atggttaata acttatttct tcggtgcttc tcctagtagt 600

gaaaagaatt tctttgaaat aaatccttta aatgacgccg taagaagcat tagaaatagt 660

aaatatggtt atagtaatga aaatgatgtt caagtatcct atagtagttt acagaactat 720

atatctgatc tttcttcgct ggtaagtaaa ggggttttat tagaagaaaa agaattttat 780

gcatctgtcc gtttaagggg cggtcctcaa gtttcagatt taaaaaatca tgggattcgt 840

tatattgaat taagaaacct agatttaaat ccttttgaaa catatggaat tagccatgaa 900

caagcagaat ttctacatct tttcctcatc tatttactct ggattgatca agacgataat 960

aatgatgaat gggtaaaaat cggtgatttt caaaataatt tagtggctct tgaacatccg 1020

ttagaacata cgcaatttaa aacagatgct gagcgtatta ttgatgagat ggaacattta 1080

acagggctgt tagacataac tgtttcgaat accttatttg ttaacttgag agaaatgcta 1140

acagatccaa gtaagacttt agctggaaga ctttataaag agattataaa aagtagtcaa 1200

agtcaggtag cttcccgtat tgctaaagaa aattataaaa aagcatggga taaaccatat 1260

caactatcag gatttactga catggagttg tctacacaaa ttttaatgtt tgatgctatt 1320

caacaaggat tacaagtaga cgttttagat cgacaagatc aatttttaaa actacagtta 1380

ggaaatcatg ttgaatatgt gaaaaatggg aatatgacaa gtaaagatag ttatatatca 1440

ccattaatta tggaaaataa aacagtaaca aagaaaattc ttcaacagca tggatttcgt 1500

gtaccaatag gtgaggaatt tagtgatata gaaaaagcct tacgttccta tgatatattt 1560

gcaggaaagc cttttgtcgt taaaccaaaa acaacaaatt atggtttagg aatatctatc 1620

tttaaagaaa acggagcaag ttacgaagac tatcaaaaag cactcacaat agcatttaaa 1680

gaggactcat cagtattaat agaagaattt attaatggaa cagaatatcg attttttgtg 1740

ctagatggca aagtttctgc cgttttatta cgaattccag ccaatgttat aggagacggt 1800

tcacatacga ttgaagagtt agtcgctcaa aagaacctga attcattaag aggaatggac 1860

catagaacac ctttagaaaa tatacaatta ggtgaattag aggtcctaat gcttaaagct 1920

caagggtatc gaaaagattc tattccaaca agtgatgaaa ttgtttttct gcgagaaaat 1980

tctaatgtca gtacaggtgg agactcaatt gatatgacag atcaaattcc cgatgattat 2040

aagaaaattg ccgtagatgc tgtgtcggca cttggagcaa atattagtgg tattgatttg 2100

attattgaaa atacagaagt tcctgcagct aataaaaatg cttatggaat tattgaggca 2160

aactttaatc catcgatgta tatgcatata tatccgtata aaggcaaatc cagacgtttg 2220

accatctgta tattacatta tttattccct gaactaccaa aaaaataa 2268

<210>11

<211>2256

<212>DNA

<213>Streptococcus sanguinis

<400>11

atgatgacga ttaatcaatt attgcagaaa ctggatacag caagccccat tctccaagcg 60

acctttggtc tggagagaga aaatctgcgg gtaacaactg acggacactt agctcaaact 120

gcccatccga gtcagctggg ttcccgcaat ttccacccga ccatccaaac agacttcagc 180

gagcaacaac tggaactaat tacacctatc gctcactcga ctaaggaagc acgacgcttg 240

ctgggagcta tcagcgatgt ggctggccgc tcgattgacc agaatgaacg cctctggccc 300

atgtccatgc caccacagct gacggaagaa gagattgcca tcgctcatct ggaaaacgac 360

tacgaacgcc actaccgaga gggtttagcg aaaaaatatg gcaaaaaact acaggccatc 420

tctggcatcc actacaacat ggagctagga aaagatctgg tcacttctct ctttcaagtc 480

agctcctacc attcactcaa ggactttaaa aatgacctct atctcaagct ggctcgaaac 540

tttctgcgtt tccgctggat tttaacctat ctctacgggg ctgccccttg ggcagaagcc 600

ggtttctaca gtcaggaaat ttcccagcct atccgctctt ttcgtaacag cgactatggc 660

tatgtcaatg acgagaatat tcaggtatcc tacgcttctt tggaacaata tataactgat 720

attgaaaact acgttcagtc aggcgagctc agcgctgaaa aagaatttta ctcagccgtt 780

cgcttccgag gacagaagca caatcacgcc tatctggagc agggtatcac ctatctggaa 840

ttccgctgtt ttgacctcaa tccttttgac catttaggca ttagccagga aaccttagat 900

actgtccatc tctttttact gagcctgctc tggctagatg atgtagaaaa tgttgatact 960

gcattaaaag ctgcccacga cttgaaccaa aaaattgctt gtagccaccc gctgactgcc 1020

ctgccagatg aggcagacag ctcagcactt ctccaagcca tggaagagct catccagcat 1080

tttgaactgc cgacatacta ccaaaccttg cttcagcaac ttaaggaggc actactaaat 1140

cctcagctaa ccctatctgg tcaactcctg cctcatatcc agcaggattc cttaatggcc 1200

tttggactag aaaaagcaga ggaatatcac cgctatgctt ggacggctcc ttatgctctt 1260

aaaggctacg aaaatatgga actgtccacc cagatgctgc tctttgatgc tattcaaaag 1320

gggctgaatg tcgacatctt agatgaaaat gaccaatttc tcaaactttg gcatggtcac 1380

catgtggagt atgtcaagaa cggcaacatg acctccaagg ataactatgt tattcctctg 1440

gctatggcca ataaaacagt caccaaaaag attttggcag aagctgactt tcccgttcca 1500

gctggagcag aattttcttc tcttgaagag ggactggcct attacccttt gattagggac 1560

cgacaaattg ttgtcaaacc taagtcaacc aacttcggac tgggcatctc catcttccaa 1620

gaaccagcta gcctggaatc ttatcgcaag gctttagaga ttgccttttc agaagacgct 1680

gctgtcttag tcgaagaatt catcgctgga acggaatacc gcttctttgt cttagacggt 1740

caatgtgagg cagtcctctt acgagtggcg gctaatgtcg ttggagacgg tcaacatacc 1800

gtgagagaat tggtggccat caaaaacgac aatcccctgc gggggcgcga tcatcgctcg 1860

ccgctcgaaa tcattgaact gggtgatatt gaactgctta tgctggatca gcaaggctat 1920

ggaccagatg atatcctgcc tgatggagtc aaggttgact tgcggcgcaa ttccaacatt 1980

tctactggcg gagactcgat tgacgtcaca gacagcatgc acccatctta taaggaactt 2040

gctgctgaca tggcaaaggc tatgggagct tgggcttgtg gcgttgacct gattatccct 2100

gacagctctg ctatttccac aaaggaaaat cccaactgta cctgcatcga gctcaacttc 2160

aacccctcta tgtatatgca cacctattgt gctgagggac cgggacaaag catcacacct 2220

aaaatactgg ccaaactttt cccagaaatg gactga 2256

<210>12

<211>2256

<212>DNA

<213>Streptococcus gordonii

<400>12

atgacgatta atcaattact gcagaaactg gatacatcaa gccctattct tcaagcgacc 60

tttggtctgg agagggaaaa tctccgagtg acgccagatg gacacttggc tcaaactgcc 120

catccgaaca agcttggttc ccgtaatttc cacccaagca tccaaacaga tttcagcgag 180

caacaactag aactaataac acctgtcgct cattcaacta aggaggcacg acgcttactg 240

ggggctatta gtgacgtggc ggcccgctcg attgatcaga acgagcgcct ctggcctctg 300

tccatgccac cgcagctgac tgaagaggag attgtcattg ctcgtctaga aaacgcctat 360

gagcgccact accgagaggg cttggcggaa aaatatggca aaaaactaca ggctatctct 420

ggtatccact acaacatgga gctagggaaa gatctggtaa ctgctctctt ccgagtcagc 480

tcccaccatt cgctaaaaga ttttaaaaat gacctctatc tcaagctagc tcgaaacttt 540

ctgcgtttcc gctggatctt aacctatctc tacggcgctg ctcctctagc agaagctggc 600

ttctacagtc aggacagttc ccagcctatc cgctcttttc gtaacagcga ctatggctat 660

gtcaatgacg agaacattaa agtttcttac gcttctttgg agcaatatgt aactgatatt 720

gaaaactacg ttcagtcagg caagctcagc gctgaaaagg aattttactc agccgttcgc 780

ttccgtggac aaaagcacaa tcgtgtctat ctggaacaag gtatcactta tctagaattc 840

cgctgcttcg atctcaatcc ttttgaccat ttaggcatta gccaagagac cttagatact 900

gtccatctct ttttactgag cctgctttgg ctagatgatg ttgaaaatgt tgatgtggca 960

ttaaaagctg cccatgactt gaaccaaaag attgcttgta gccatccgct aactgccctg 1020

ccagatgagg cagacagctc aacactgctc caagccatgg aagagctcat ccagcacttt 1080

gaactgccaa cttactacca aagcttgctt gagcagctta aagaatcact tctgaatcct 1140

cagctaaccc tatctggtca gctcctgcct catatccagc aggattcctt aatggacttt 1200

ggattagaaa aagcagagga gtatcaccac tatgcttggt cggctcctta tgctctcaaa 1260

ggctacgaga atatggaatt atctactcaa atgctactct ttgatgccat acaaagaggt 1320

ctaaattttg aaattttaga tgaaaacgat caatttctca agctttggta tggtccccat 1380

gtggagtatg tcaagaacgg caatatgacc tccaaggata actatgtcat ccctctggcc 1440

atggccaata aaatagtcac aaaaaagatt ttggctgcag ctgactttcc tgttcctgct 1500

ggagcagaat ttaccacact tgaagagggg atactctact atcctttgat taaagaccga 1560

cagatcgttg tcaaacccaa gtcaaccaac ttcggactgg gcatctccat cttccaagaa 1620

ccagctagcc tggaagctta tcgcaaggct ttggagattg ctttttcaga agacaccgcc 1680

gttttggtgg aggaatttat cgcaggaacg gagtaccgat tctttgtctt agatggtcag 1740

tgtgaggcag tcctcttgcg agtagcagcc aatgtcgtcg gagacggtca gcataccgtg 1800

agagaattag ttgccatcaa aaacgacaat cccctgcgcg gtcgagacca tcgttcaccg 1860

ctcgaaatca tcgaactggg cgacattgaa ctgctcatgc tggaccagca aggttatgga 1920

ccagacgata tcctgcctgc tggagtcaag gttgacttgc gacgcaattc caatatttct 1980

accggcggag actcgattga tgtcacagac agcatgcacc catcttataa ggaactcgct 2040

gcggacatgg caaaggctat gggagcttgg gcttgtggcg ttgacctgat catccctgac 2100

agctctgcta tttctaccaa ggaaaatccc aactgcacct gcattgagct caatttcaac 2160

ccctctatgt atatgcatac ctattgtgct gaggggccgg gacaaagcat cacccctaaa 2220

atactggcta aacttttccc agaaatggac ctgtaa 2256

<210>13

<211>2262

<212>DNA

<213>Streptococcus uberis

<400>13

atgcttaatc agctcttaca aaaactccct caaaatacag acattttgga ggcaactttc 60

ggcttggaac gtgaaggcct tcgcctaaca caagaaggga cactggcaca aacagaccac 120

cccaaagccc ttggttcacg aagcttccac ccttatatcc aaaccgactt tagtgagcaa 180

caactggaat tgattacccc catttctcag tcgacacagg aggctagaag acgcctaggc 240

gccatctttg atgtcgcaca gcgtagcctt gaagaagacc aggtcatctg gccactttcc 300

atacccccct atatcgaaga agatcagatt cagattgcta aactggacaa agccgaagag 360

gttcgctacc gggaacaact cgcccaaact tatggcaaaa aacttcaatc catttcaggc 420

atccattaca acatagaact aggtaaaaac ttaacccaaa acctgtttgc ccttagcgac 480

caagaagacc ttaaagactt caaaaatgcc atctacatga agttagctcg ccaattcctc 540

aactaccaat ggctggttac ctaccttttt ggcgctagcc cactagctga aaaagacttc 600

taccctcaga tgccaacaga gctcgtccgt tccttaagag ctagccgccg ttacggatac 660

agcaaccacg aggaattgtc catttccttc tcctcccttg aaaactatgt caaggatatg 720

gaaaatgcct tggccaccgg tatcctgtcc ctagaaaaag aattctactc cccagtccgc 780

ctccgcggca gcaaacatag ccgtgactac ttgtcagaag gcatcaccta cctagaattc 840

cgcaacttcg acatcaaccc cttcgacaag ctaggcatca gccaaaaaac cttagacagc 900

ttccacctct tcctcctcag cctcctatgg ctagacgacc taaaagacag cgaccaagaa 960

ctgacaaaag cccgtcaaat caacgaagaa gtggccttag cccaccccat gagcccactg 1020

cctgacccag aactggcaag accggtcctc aaggccatga cagaactaat ccaacacttc 1080

ggtttagatg cttactacca agacttggtg gcagacctag aactagctat caaggaacct 1140

gaaagaacca tctctggaca cctttttggc aaaatcgcta acgcttcttt ggctgccttt 1200

ggcttagacc aagcaaaaac ttaccaccaa gaggctaccc aggctcccta cgcactcaaa 1260

ggttatgaaa gcatggagtt gtctacgcaa atgctcatgt tcgatgccat ccaaaagggg 1320

cttcatctgg acatcctgga tgaaaatgac caatttttga agatctggca cggcgaccat 1380

gtcgagtatg tcaaaaacgg caacatgacc tcacgtgaca actacgtggt tcctctggcc 1440

atggccaaca aaacggtgac caagaaaatc ttggatcagg ctggttttcc aactccgaag 1500

gggcaggaat ttgccagcaa agaccaggct atccgttact tcaatcagat agcggacaag 1560

gccattgtgg tcaagcctaa atcaaccaac tttggactgg gcatttccat cttccaaaag 1620

ccggccagtc aagctgacta tgaaaaagcc gtggaaattg cctttgcgga agaccgcgac 1680

atcttggtgg aagaatttat tgccggtact gaataccgct tcttcacctt gaatggcaag 1740

tgtgaggccg ttttactgcg tttaccggct aatgtggtgg gcgacggtat ccatacggtc 1800

aaagaattga ttgacctcaa aaatcaaaac ccacttcgag gactagacca ccgttcaccc 1860

ttagagaaaa tccaattagg tgatattgaa aaactgatgt tagctcaaga gggatacggt 1920

cccgactccg tcttagccaa aggggttaag gtcgaacttc gccgcaactc caacatttca 1980

actgggggcg actcggttga tatgaccgat gagatggacc cttcttacaa agcattagca 2040

gctcaaatgg cggatgccat gggagcctgg gtctgtggtg tggacctgat tattcctgac 2100

ccaagtcaaa aggctagtct tgaagagcct aactgccatt gcattgagct caacttcaac 2160

ccttccatgt acatgcatac ttactgtcag gaagggcccg gccaagccat cacccctaaa 2220

attttacaag ccctcttccc agagttgcct atttatccat ag 2262

<210>14

<211>2265

<212>DNA

<213>Streptococcus thermophilus

<400>14

atgacattaa accaacttct tcaaaaactg gaagctacca gccctattct ccaagctaat 60

tttggaatcg agcgcgagag tctacgtgtc gataggcaag gacaactggt gcatacacct 120

cacccatcct gtctaggagc tcgtagtttc cacccctata ttcagactga tttttgcgag 180

tttcagatgg aactcatcac accagttgcc aaatctacta ctgaggctcg ccgatttctg 240

ggagctatta ctgatgtagc tggccgctct attgctacag acgaggttct ctggccttta 300

tccatgccac ctcgtctaaa ggcagaggag attcaagttg ctcaactgga aaatgacttc 360

gaacgccatt atcgtaacta tttggctgaa aaatacggaa ctaaactaca agctatctca 420

ggtatccact ataatatgga actgggtaaa gatttagttg aggccttgtt ccaagaaagt 480

ggtcagaccg atatgattgc cttcaaaaac gccctctatc ttaagctggc tcagaactac 540

ttgcgctacc gttgggtgat tacctatctc tttggggcct cacccatcgc cgaacaaggt 600

ttctttgacc aggaagttcc agaacctgtg cgttccttcc gtaacagtga ccacggctat 660

gtcaataagg aagagattca agtatccttt gtaagtctag aagattatgt ctcagccatt 720

gaaacctata tcgaacaagg agatttgaat gcagagaaag aattttactc agctgttcgt 780

ttccgtggac aaaaggttaa tcgttccttc cttgacaaag gaatcaccta cctagagttc 840

cgtaatttcg accttaaccc ttttgagcgt atcggtatta gtcagactac tatggacact 900

gtgcacttac tcattttagc cttcctttgg cttgatagcc ctgaaaatgt cgaccaagct 960

cttgcacaag gccacgcgtt aaatgagaaa attgccctct ctcatcctct agaacctcta 1020

ccttcggagg ctaaaactca ggacattgta actgccctag accaactggt gcaacacttt 1080

ggacttggtg actatcatca agatctggtt aagcaagtta aggcagcctt tgcggatcca 1140

aatcaaacgc tctctgccca gctcttaccc tatatcaaag acaaatctct agccgaattt 1200

gctttaaaca aggctcttgc ctatcatgat tacgactgga ctgcccacta tgctctcaag 1260

ggctatgaag agatggaact ctccacccag atgttgctct ttgatgccat ccaaaagggg 1320

attcactttg aaatattgga tgagcaagat caattcctaa aactttggca ccaagaccat 1380

gttgaatacg tcaaaaacgg taacatgacc tcaaaagaca actacgtggt tccccttgct 1440

atggctaata agaccgtaac caagaagatt ctagcagatg ctggctttcc agttccttca 1500

ggagacgaat ttaccagtct tgaggaagga cttgcctact accctcttat caaggataag 1560

caaattgttg tcaaacccaa gtcaactaac tttggtctgg gaatttccat tttccaagaa 1620

cctgccagtc ttgacaacta tcaaaaagcc cttgaaattg ctttcgcaga agatacctct 1680

gtccttgttg aagaatttat tccaggaacc gaataccgtt tcttcatctt ggatgggcgt 1740

tgtgaggctg ttcttctgcg tgtcgctgcc aatgttattg gtgatggcaa acacaccatt 1800

cgtgaactag tcgctcagaa aaatgctaat ccattgcgtg gccgtgatca ccggtcacct 1860

ctggaaatca ttgagctagg agacatcgaa caactaatgt tagctcaaca gggttacaca 1920

cctgatgata ttctcccaga aggaaaaaag gtcaatctgc gtcgtaattc caacatctct 1980

acaggtggtg actctattga tatcactgag accatggatt cctcttacca agaattagcc 2040

gcagccatgg caactagcat gggcgcctgg gcttgcgggg ttgatctgat aattccagat 2100

gaaactcaaa ttgccaccaa ggaaaatcct cattgcacct gcattgagct caactttaac 2160

ccttcgatgt atatgcacac ctactgtgct gagggtcctg gccaagctat cactactaaa 2220

atcctagata aactttttcc agaaatagtg gctggtcaaa cttaa 2265

Claims (7)

1. a difunctional glutathione synthetase has two kinds of functions of gamma-glutamylcysteine synthetic enzyme and glutathione synthetase simultaneously, and it is characterized in that: the aminoacid sequence of described difunctional glutathione synthetase is shown in SEQ ID NO:1.

2. difunctional glutathione synthetase according to claim 1, it is characterized in that: described difunctional glutathione synthetase is encoded by gshF, and its gene order is shown in SEQ ID NO:8.

3. method of utilizing difunctional glutathione synthetase synthesizing glutathion claimed in claim 1 is characterized in that: be the catalyzer synthesizing glutathion by the genetic engineering bacterium of expressing actinobacillus pleuropneumoniae (Actinobacillus pleuropneumonia) gshF; Or the difunctional glutathione synthetase that is extracted by the genetic engineering bacterium of expressing actinobacillus pleuropneumoniae (Actinobacillus pleuropneumonia) gshF is the catalyzer synthesizing glutathion, and the aminoacid sequence of described actinobacillus pleuropneumoniae (Actinobacillus pleuropneumonia) gshF is shown in SEQ ID NO:1.

4. the method for synthesizing glutathion according to claim 3 is characterized in that: utilize efficient promoter to regulate and control described actinobacillus pleuropneumoniae (Actinobacillus pleuropneumonia) gshF genetic transcription.

5. the method for synthesizing glutathion according to claim 3, it is characterized in that: the Host Strains of described genetic engineering bacterium is bacterium, yeast or mould.

6. the method for synthesizing glutathion according to claim 5, it is characterized in that: described bacterium is intestinal bacteria (Escherichia coli), Bacillus subtilus (Bacillus subtilis), in the pseudomonas (Pseudomonas) one or more, described yeast is yeast saccharomyces cerevisiae (Saccharomyces cerevisiae),), pichia pastoris phaff (Pichia pastoris), in the multiple-shaped nuohan inferior yeast (Hansenula polymorpha) one or more, described mould are aspergillus oryzae (Aspergillus oryzae) or aspergillus niger (Aspergillus niger).

7. method of utilizing difunctional glutathione synthetase synthesizing glutathion claimed in claim 1, it is characterized in that: the genetic engineering bacterium fermentation by culture expression actinobacillus pleuropneumoniae (Actinobacillus pleuropneumonia) gshF produces, and the aminoacid sequence of described actinobacillus pleuropneumoniae (Actinobacillus pleuropneumonia) gshF is shown in SEQ ID NO:1.

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