JPH05508997A - Novel thermostable pullulanase - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Novel thermostable pullulanase Technical field The present invention is in the field of thermostable enzymes. More specifically, the present invention relates to Pyrococcus Novel thermostable pullulanase obtained from strains belonging to the genus and methods for producing these enzymes.

Furthermore, the present invention relates to a strain belonging to the genus Pyrococcus which is substantially composed of pullulanase components of the same species. Produced by recombinant DNA encoding pullulanase derived from the genome of a bacterial strain The present invention relates to a pullulanase preparation prepared by the present invention.

Furthermore, the present invention provides for the conversion of starch by pullulanase, as well as for liquefaction and/or saccharification. Wait for use in the process.

Background technology Thermostable pullulanase is, for example, Bacillus acidoplulyticus (Bacillus acidoplulyticus). llus ac (formerly known as "Koshi Tsujie Tadashi"), and was used in the industrial saccharification process. Their use is known (see European Patent Application No. 63,909) , extensive research has been carried out to meet the demand for more thermostable enzymes. 0 An object of the present invention is to provide a novel pullulanase with improved thermostability. Ru.

Summary of the invention A new strain belonging to the genus Pyrococcus exhibits unique thermostability and thermal activity. It has now been discovered that this enzyme produces a pullulan-degrading enzyme.

Therefore, in the first aspect of the invention, Pyroconcus boisei (h animals ■ w oesei) (DSM No, 3773) or Pyrococcus furiosus (h Pullulanase derived from two pairs of animals (■furiosus) (DSM No. 3638) Provided are pullulanases that exhibit immunochemical properties equivalent or partially equivalent to those of the present invention.

In another aspect, the present invention provides a pH 1 substrate and calcium deficiencies optimized for pH 5-7. The residual activity, when measured after incubation in the presence of exceeded 90% after 20 minutes at 110°C, and exceeded 30% after 20 minutes at 110°C, and 85-115 Provided is a pullulanase characterized by having an optimum temperature at °C.

In a third aspect, the present invention provides a pullulanase-producing strain of Pyrococcus with a carbon source, nitrogen The process involves culturing in a nutrient medium containing sources and inorganic salts, and then recovering the desired enzyme. Provided is a method for producing pullulanase of the present invention, comprising:

In a fourth aspect, the present invention provides a protein derived from the genome of a strain belonging to the genus Pyrococcus. The same species of pullulanase produced by recombinant DNA encoding DNA Provided is a pullulanase preparation consisting essentially of minutes. In a more specific aspect, the book The invention is based on Pyrococcus boisei (DSM No. 3773) or Pyrococcus boisei ・Immunochemically identical to pullulanase derived from Furiosus (DSM No. 3637) consisting of substantially the same type of pullulanase components having similar or partially equivalent properties. A pullulanase preparation is provided.

In a fifth eleventh aspect, the present invention provides for isolating a DNA fragment encoding said pullulanase. The step of inserting this DNA fragment into an appropriate plasmid vector with an appropriate expression signal. This plasmid vector is autonomously replicated into a suitable host. The process of introducing this host microorganism as a plasmid or integrated into the chromosome. A step of culturing an organism under conditions that induce the expression of burlanase, and Provided is a method for producing enzymes with high expression levels, which comprises a step of recovering pullulanase.

In a sixth aspect, the invention provides the use of a pullulanase of the invention in a starch conversion process. directed towards. In a more specific manner, the present invention provides a pullulanase of the present invention and a group of from lucoamylase, α-glucosidase, β-amylase or other saccharifying enzymes Including the step of saccharifying starch hydrolyzate in the presence of one or more selected enzymes. A method for converting starch into glucose- and/or maltose-containing syrup is proposed. provide In another embodiment, the present invention provides a pullulanase of the present invention and a thermostable α - Simultaneous liquefaction/debranching (technical cutting) in the presence of amylase, followed by In addition to pullulanase, glucoamylase, α-glucosidase, β- Saccharification in the presence of an enzyme of IN or higher selected from amylase or other saccharification enzymes Conversion of glucose and/or maltose content of starch to syrup provide a method for

Brief description of the drawing The invention will be further described with reference to the accompanying drawings.

Figure 1 shows the relationship between temperature and enzyme activity of the enzyme of the present invention, Figure 2 shows the relationship between temperature and enzyme activity of the enzyme of the present invention. Figure 3 shows the relationship between enzyme pH and enzyme activity at various temperatures (・100°C, 110°C and 1120°C) shows changes in enzyme activity of the enzyme of the present invention over time. , and FIG. 4 shows the restriction enzyme map of plasmid psJ933.

Detailed disclosure of the invention mouse element The present invention provides a novel pullulan-degrading method that can be obtained from a strain belonging to the genus Pyrococcus. Enzymes or mutants or variants thereof or obtained from a strain of Pyrococcus A pullulanase with immunochemical properties equivalent or partially equivalent to that of pullulanase. Concerning orchid degrading enzymes.

Enzyme variants or mutant enzymes are changes in the DNA nucleotide sequence of the original gene. means an enzyme or a derivative thereof that can be obtained by conversion. Is there a variant of the enzyme? Alternatively, a mutant enzyme can be produced by injecting the DNA nucleotide sequence encoding the enzyme into a suitable host microorganism. can be expressed and produced when inserted into a suitable vector in an organism . The host microorganism need not be the same microorganism from which the original gene was obtained.

The pullulan-degrading enzyme of the present invention is specified by the following characteristics.

You've got a lot of blood The enzymes of the invention are active over a very wide temperature and pH range. These enzymes are 4 Shows pullulan decomposition activity in the temperature range from 0℃ to over 130℃, 85-115℃ More specifically, these enzymes exhibit an optimum temperature in the range of 100 to 110°C. It has pullulan-degrading activity in the pi range from pH 3.5 to over 8; Specifically, the optimum pH is in the range of pH 5.5 to pH 6.5, and at pH 8. Approximately 55% pullulan degrading activity can be measured. After 4 hours at 100°C, these pullulanase does not substantially lose its pullulan-degrading activity when heated at 100°C for 24 hours. Afterwards, a residual activity of 65% can be determined. Approximately 10% residual activity after 1 hour at 110°C can be measured. Addition of metal ions is not necessary for catalytic activity. The enzyme is Inhibited by clodextrin and β-cyclodextrin.

The particular enzymes tested were the α-1°6-glycosides of pullulan in an irregular manner. OP3 by acting on the bond. OP6 and OP9 (OP: degree of polymerization) are produced. low Branched oligomers of molecules are also affected by these pullulanases. On the other hand, Dextra pullulanase derived from other bacteria that is not hydrolyzed by the pullulanase of the present invention. Differently, these pullulanases bind α-1,6-linkages of small substrates such as panose. can also be hydrolyzed (products are maltose and glucose).

Nine skins on the top The pullulanase of the present invention is derived from Pyrococcus boisei (h individuals ccus). It has the immunochemical properties of

These immunochemical properties can be determined by immunological cross-reactivity identification tests. This same The specific test was conducted by N. H. Axelsen, Handbook of Immuno -precipitation-in-Gel Techniques (Bl ackwell 5 scientific Publications, 1983 ) Well-known ophthalony according to Chapters 5 and 14 - double immunodiffusion or double-headed crossover This can be done by immunoelectrophoresis. “Antigen equivalence” and “r-partial antigen equivalence” The words are listed in Chapters 5, 19 and 20 of the same book.

By sensitizing rabbits with purified pullulanase of the present invention according to the above method, single Specific antiserum is produced. The immunogen is mixed with Freund's adjuvant and then Rabbits were injected subcutaneously every second week.

Antisera were obtained after total sensitization for 81 days and then described in N. H. Axelsen, supra. Immunoglobulin was prepared as described.

Blue Ze's Meeting 11 The pullulanase of the present invention can be prepared in a suitable nutrient medium containing a carbon source, a nitrogen source and an inorganic salt. Cultivate a pullulanase-producing strain belonging to Pyrococcus and then collect the desired enzyme. It can be manufactured by

P-woesei (P, woesei) and P-furiosus (P, furio) sus) is a typical seedling of the genus Pyrococcus. Representative strains of P. boisei is available from DSM (as No. 3773) and written by P. Furiosus. Representative strains are available from DSM (as No. 3638).

Microorganisms belonging to the hyperthermophilic archaeon Pyrococcus have a pH of 80 to 105 °C (14 , 5 and 7.5, and starch, glycogen, and dextrose. It can grow on various complex media containing phosphorus and oligosaccharides. The life of these bacteria It is clear that significant thermoactive intracellular and extracellular pullulanases are produced during growth. Became.

Continuous feeding of nutrient medium for the purpose of establishing a fermentation method with good industrial applicability A method for producing pullulanase has been developed. Continuous supply of nutrient medium stimulates enzyme production It was found that promoting Moreover, the more limited use for growing Pyrococcus spp. A new nutrient medium was developed.

The difference from other media described is that this medium is not cloudy and does not contain elemental sulfur. That's a good thing. Optimum growth and enzyme production in this medium are based on N, /Co, or N2 This can be achieved by continuously replenishing.

Specific examples of such medium compositions are shown in Tables 2 and 3.

1-12 Complex nutrient medium containing elemental sulfur-1 (per liter): sea salt (llieg and, Krefeld, FRG) 30 gKHzPO41,5g NiC1□X 6Hz0 2mg This trace element solution (see below) 10ml 1 sulfur yellow (powder) 10g Yeast extract 1g Peptone 2g Starch 2g Resazurin 1mg NazSX91 (zo 500mg Adjust pH to 6.3-6.5 Temperature 90-100°C Hz/co□ supply: 80/20 Umbrella trace element solution (per liter): Titriplex I (Merck) 1.5 g MgSOa X 7Hz0 3.0g Mn5On X 2L0 5005g NaC11g FeSO,X 7M, 0 100*g CaCIz　X　2Hz0　10011g100l1　X　7HzO180mg Cu5Oa X 5HzOLong KAI (504) t 10mg H Go BO Go 10-g NazMoOa X 2Hz0 10mg to ich X 6Hz0 2511g CaC1z　X　2H! 0 10mg Adjust pH to 7.0 1-also Complex nutrient medium that does not contain elemental sulfur - 1f (per liter): (NH Mountain SOa) 1.3g Mg5Oa　X　7Hz0　250*gFeSO,X　7JO38gg NazSeOs　X　5Hz0　5　tt　M umbrella trace element solution (see below) I Omj Cystine x HCI 500mg Adjust pH to 6.2-6.5 Temperature 90~lOO°C Nz/Cot (7) supply: 80/20 trace element solution (per 1 liter): MnC1z　X　4Hz0　100mgCo(:lx　X　6Hz0　200m gNiCl, X 6H1O100wg ZnC1z 100mg CaC1z　X　2HzO50+5g Cu5Oa X 2Hz0 50mg NazMoO4X 2Hz0 5011gl-main Complex nutrient medium without elemental sulfur - Ill (per 1 liter): (NH4) t sOa　1.3　g FIgSOa X 7HtO250mgNaC13Q, Og KHtPOa 1.4 g CaCl, 50mg Fe5Oa　X　ll1tO38mgNatSeOs　X　5HzO5u　M Umbrella trace element solution (see below) lhl Tryptone 1g Enzyme extract 1g Starch 1g Resazurin 11g Cystine X HCI 50g Adjust p[I to 6.2-6.5 Temperature 90-100℃ Supply of Nt/Co: 80/20 Trace element solution (per liter): MnC1* X 4Hz0 100mg CoCIx X 6Ht0 200mg N1Ch　X　6)1i0　100gZnCIg　100mg CaC1t X 2H! 0 50mg CuSO4X 2Hz0 50mg NazMoOa X 2HtO50mg1 Testing of strains belonging to Pyrococcus has shown that they produce small amounts of pullulanase. production by culture of these microorganisms necessarily has very low yields, since . Moreover, studies using enzymes from these microorganisms have shown that they are Preparations obtained from these microorganisms to produce complex mixtures of lanase components has been shown to have relatively low specific activity.

These facts hinder the practical development of production methods by culturing Pyrococcus strains. was. Low yields and the difficulty of optimizing the production of single components in multienzyme systems have led to making it difficult to carry out effective production at industrial costs of lanase preparations, and Their practical use is due to the fact that it is necessary to use a large amount of enzyme to obtain the desired enzymatic action. have been hampered by difficulties.

These shortcomings allow for the production of single-component enzyme preparations with high specific activity. Potential improvements can be made by using high-yield methods. Therefore, the present invention A further object is to produce recombinant pullulanase components comprising substantially homologous pullulanase components. and a high expression method for producing this single-component preparation. There is a particular thing.

Emperor:” (minute ILt declaration) The present invention provides recombinantly produced pullulanase components comprising substantially the same species of pullulanase components. Provide a lanase preparation. The DNA encoding this pullulanase component is It can be derived from any microorganism belonging to the genus Kotsucus. Preferably, pullulanar The DNA encoding the enzyme component was obtained from one strain of P. boisei or P. furiosus. be guided. The representative strain of P. boisei is DSM (No. 3773). Representative strains of P. furiosus are available from the Institute (No. 36 Available from 38).

The pullulanase preparation of the present invention can be used by P. boisei (DSM No. 3773) or is the same as pullulanase derived from P. furiosus (DSM No. 3638). or partially equivalent immunochemical properties (e.g., N, H, It consists essentially of pullulanase components of the same species exhibiting a

Preferably, the pullulanase preparations of the invention contain at least 6 Burulanase units (pu), more preferably at least per gram of protein It has a pullulan degrading activity of 20 PU. Measuring method of pullulan degrading activity and determination of pu The definition is described in Example 8.

The pullulanase preparations of the present invention are single component preparations. Furthermore, the puller of the present invention The nase component exhibits an apparent molecular weight of 95 k[l; these characteristics are Measured in Example 9.

The pullulanase preparation of the invention has a pH of 4, when measured as in Example 7. It has an optimum pH of 5 to 6.0, more specifically 4.5 to 5.5.

The pullulanase preparation of the present invention, when measured as in Example 7, It has an optimum temperature of 15°C, more specifically 95-115°C.

The pullulanase preparation of the present invention, when measured as in Example 7, After 30 minutes at °C, the residual activity exceeds 80%, preferably exceeds 90%. more than 70%, preferably more than 80% residual activity after 2 hours at °C, and more than 60%, preferably more than 70%, even more preferably more than 70% after 4 hours at 100°C. Preferably, it exhibits thermal stability with a residual activity of greater than 80%.

Studies regarding substrate specificity have shown that the pullulanase preparations of the present invention are nearly selective for pullulan. selectively decomposes panose into maltotriose, and almost selectively decomposes panose into glucose and maltotriose. and convert all soluble starch into glucose oligomers, i.e. glucose It separates hydrolysis products that are converted into lucose, maltose, glucotriose, etc. The rate of hydrolysis of pullulan is significantly faster.

Brunase Recombinant DNA methods were used to obtain pullulanase as a single-component preparation. The method of the invention preferably involves the isolation of a DNA fragment encoding pullulanase; Combining the DNA fragment with an appropriate expression signal in an appropriate plasmid vector , integration of the plasmid vector into an autonomously replicating plasmid or chromosome introduction into a suitable host as either a microorganism, and expression of pullulanase in that host microorganism. and the recovery of pullulanase from the culture. This is a high expression method.

Recombinant DNA techniques and methods are known in the art and are within the skill of the art. This would be easier to implement.

The pullulanase component of the pullulanase preparation of the present invention is derived from Pyrococcus sp. Therefore, production is possible. Preferred species are P. poisei and P. furiosus. Ru. A DNA fragment encoding a pullulanase component or its precursor can be, for example, a pullulanase component or a precursor thereof. Lulanase-producing microorganisms [e.g., P. boisei (DSM No. 3773) or is P. Furiosus (DSM No.

3638))) was established, and then a pullulana Oligonucleotides synthesized based on the entire or partial amino acid sequence of Hybridization to cleotide probe or appropriate pullulan degrading activity Selection of expression clones or reactivity with antibodies against natural pullulanase components By selecting positive clones by standard methods such as selecting protein-producing clones, isolated.

Screening for suitable NA sequences and construction of vectors are well known in the art. It can be performed using standard methods.

A suitable promoter, driver, that allows expression of pullulanase in a specific host microorganism Appropriate replication comprising a pelleter and terminator sequence and an origin of replication Once the above-selected DNA sequence is inserted into a possible expression vector, the corresponding host The vector becomes able to reproduce in the main microorganism.

Next, with the obtained expression vector, Escherichia coli (Escherichia coli) Namitan), Bacillus, Aspergillus (Hadan I...) and is a suitable host cell such as a microorganism belonging to the genus Streptomyces. can be transformed. Using E. coli as the host microorganism suitable plasmids include pBR322 and pACYC or It is a derivative of Bacillus species were used as host microorganisms. suitable plasmids are pUBllo, pc194 or pE1. 94, a suitable Bacillus species is B. subtilis (B, 5ubti lis), B. licheniformis (B.

1icheniforwis), B-amyloliquefaciens (B, adi+Io li uefaciens) or B length (B, 1entus). can be done.

The medium used to culture the transformed host is suitable for growing the corresponding cells. It can be any conventional medium suitable for use.

Other growth conditions can be selected according to principles known in the art.

Due to the thermostability of the pullulanase component obtained by the method of the present invention, the pullulanase component of the present invention It has been discovered that lanase can be purified using a very unusual method. Therefore, preferred In the case of Mr. B, the present invention boils the fermentation broth to cause a similar denaturation of natural proteins. straining, then centrifuging the fermentation process and recovering pullulanase from the supernatant. A method for purifying expressed pullulanase is provided.

By heating and boiling the culture, natural proteins, that is, the pullulana of the present invention, can be extracted. Proteins and polypeptides derived from host microorganisms other than enzyme components are denatured. It will be done. However, since the pullulanase component is a highly thermostable enzyme, such Easily tolerates processing.

Natural proteins denature within seconds, especially minutes (e.g. after 2-5 minutes). However, the pullulanase component of the present invention is stable to boiling for several hours (as in Example 7). (see Measurement of Thermostability), thus eliminating intracellular proteins associated with cell lysis. Most proteins present in humid cultures can be easily separated by centrifugation. and the purification procedure was complete with respect to the activity of the enzymatic activity (i.e., the recombinant The recombinant pullulanase was recovered without other contaminating enzyme activities).

lees Ω five stop treatment Due to the substrate specificity and high thermal stability in the absence of metal ions, the pullulana of the present invention enzymes are used in combination with other enzymes for the industrial conversion of starch to various sugars. suitable for Pullulanase from Pyrococcus, a debranching enzyme, has a thermostable α - under saccharification conditions where a significant part of their activity remains after liquefaction of starch by amylases It is particularly advantageous for use in the saccharification of α-amylase activity is present during saccharification In some cases, the accumulation of low-molecular-weight branched oligosaccharides reduces the final yield in the saccharification process. Because it does. Pullulanar with sufficient thermal stability as previously known in the art Fromozyme showed no activity against those low-molecular substances (Fromozyme (Trademark), Novo Nordisk).

The saccharification treatment is substantially as described in European Patent Application No. 63.909. It can be carried out by conventional methods such as those described above. The preferred enzyme dosage is Lulanase is in the range of 1 to 100 μg, more preferably 1 to 20 μg per 1 g of dry matter. be.

In addition, the highly thermostable pullulanase of the present invention can be used in simultaneous liquefaction/debranching treatment. This enzyme activity (alpha-amylose content), which is not common, makes them suitable for use. The advantage of the combination of degradability and pullulan degradability is that the fluid viscosity of the starch slurry can be further reduced. It is possible to increase the starch concentration during the saccharification process by achieving a reduction in It is.

The preferred enzyme dosage for simultaneous liquefaction/departitioning treatment is 1 to 1 pullulanase per gram of dry matter. 100 μg, more preferably in the range of 1 to 20 μg per 1 g of dry matter. other conditions by conventional liquefaction and saccharification processes (e.g., U.S. Pat. No. 3,912 ．． 590, European Patent Application Publication No. 252.730 and European Patent Application No. 63,909 (see also International Patent Application No. 90/11352).

The following examples are provided to further specifically explain the present invention.

Example I P. Boisei and P. Fuosas's volume P. Boisei (DSM No. 377) 3) and P. furiosus (DSM No. 3638) at 300 L each. Cultured in a stainless steel fermenter using complex nutrient medium-III listed in Table 3. . Cells were harvested after 14-24 hours of production at 90°C. Approximately 70-120g (wet weight) of cells were obtained from 30OL of cell suspension.

In addition to the intracellular enzyme, pullulanase activity can also be detected in the culture medium.

No significant difference was found between intracellular and extracellular pullulanase. . The supernatant was S reduced using a 20,000 cutoff membrane.

Cells were mixed with 50 sL of 5011M acetate buffer (pH 6,0) and then pressure Cells were disrupted in a French press at 8 MPa, then the suspension was incubated at 37 °C for 3 h. Incubation with DNase and RNase. Enzyme cell residue The zero order was separated from the containing supernatant by centrifugation at 12.0 OOX g for 20 minutes. , an enzyme sample containing both pullulanase and amylase was dialyzed against the above buffer. a Mirase activity is irreversible in the absence of its substrate (starch or dextrin) After this step, amylase activity gradually decreased as it was inactivated. After this process The sample (60 sL) contained approximately 102 U of pullulanase (17000/L) and 59 U of pullulanase (17000/L). It contained amylose degrading activity (990 U/L). Therefore, the specific activity of the enzyme is Pullulanase was 0.07 U/mg and amylase was 0.04 U/-g. acid The presence of these enzymes had no effect on the activity of these enzymes.

Bruno's A Say Pullulanase activity was measured by the following procedure. 0.5% pullulan and 50mM sodium acetate 50 μL of the enzyme sample was added to 200 μL of a solution containing Lithium (pH 6.0). mixture The articles were ink-baked at 100°C for 5.15 or 30 minutes.

Incubation was stopped by transferring the mixture to an ice/water bath (0°C).

After adding an additional 250 μL of Reagent A (see below), the mixture was incubated at 100’C for 5 min. Incubation run. Add 2.5 sL of deionized water and at 546 nIl. Absorbance was measured.

Wipe 1Δ: 3.5-dinitrosalicylic acid 1.0g2N NaOH10sL Tartaric acid, Na salt X ozo 30. Og deionized water 100L One unit (U) is measured against maltose as a standard under conditions as above. It is defined as the amount of enzyme that releases 1 micromole of reducing sugar per minute.

α-amylose” α-amylase activity was measured by the following procedure. Sodium acetate containing 1% (W/V) starch Add 100 μL or less of enzyme solution to 250 μL of um buffer (pH 5,0), Incubation continued for 1.5 hours at °C. The α-amylase activity of IU is The amount of enzyme that releases 1 micromole of reducing sugar per minute from maltose as a Defined.

Example 2 blue-ze's - Cef Loose Chroma Goofy: Extract 20 from which cells were removed obtained in Example 1 sL (46 g of protein) was transferred to a Q-Sepharose fast flow column. The protein was added to a 205M Tris-HCl buffer (pH 8,0) and NaCl gradient (0-500mM) at a flow rate of 3-17 min. eluted. A percentage of 10 sL was collected. The main fraction containing pullulan-degrading activity is Eluted at 360-400mM NaCl. In addition, the secondary peak is 470-485 Eluted with 5M NaCl. After repeating 3 times, both components containing pullulan degrading activity was collected and concentrated using a 10,000 roa cut-off membrane (1.4+sg/m 16.5 sL), the data in Table 4 was used as a reference sample for the MonoQ column (16.5 sL). FPLC: HR515) at a rate of 1 sL per minute. 2- per l experiment L was used. The buffer used for equilibration and elution was 20mM potassium phosphate (p u 7.0), both components containing pullulan degrading activity were treated with NaCl glaze (200-500mM) was used before elution. The main fraction is 360-420 Eluted with 5M NaCl. After 8 experiments, the fraction containing pullulan degrading activity was collected and then -a-condensed. After this step, pullulanase was purified 15 times. See data in Table 4.

Animals treated with Edo: Samples concentrated in the previous step (200 μL each, 680 μg/L intestine) , subjected to gel filtration using a Superose 12 column. . The buffer used was 50i+M sodium acetate (100mM NaCl, pH 5 , 5), the protein was eluted at a flow rate of 0.4 mL/min (see Table 4). thing.

Table - ■ Made by 1 fire nana Cell extraction 60 1380 102 0.07 1 100Q-Sepharose 16.5 23.0 8.0 0.34 5 7.8 Mono Q 1.5 1.1 1.1 1.06 15 1.0 Super Rose 12 11 0.0? 0.45 6.30 90' 0.4 50a+M sodium acetate buffer e50 for 10g of cells a+L was added and the cells were disrupted using a French press.

Example 3 1EJii 1-2 T’-t+? ;Nia’zb--2”41L1 to 1 degree Enzyme activity was measured between 40 and 130°C. The buffer used was 50mM acetic acid. Sodium (pH 5.5), enzyme assays were performed for 5 and 15 minutes. Ta. The results are shown in Figure 1.

The enzyme has pullulanase activity over a temperature range of 40 to 130°C, with optimal The temperature ranges from 85 to 115°C, more specifically from 100 to 110°C.

1”1 side Enzyme activity was measured between pH 3.5 and 8.0. The buffers used were It contained 505M potassium acetate, potassium phosphate and Tris. The assay is 95°C for 5 and 15 minutes. The results are shown in Figure 2.

These enzymes have pullulan-degrading activity in the pH range from 3.5 to above 8. , the optimum pH was shown at pH 5 to 7, more specifically at pH 5.5 to 6.5. pH 8, a pullulan degrading activity of approximately 55% is measurable.

Jikushuyuu These enzymes were incubated with substrate and metal in 50mM sodium acetate buffer (pH 5.5). Incubate without adding ions. Incubate at 100℃ for 6 hours No decrease in enzyme activity was observed after lysis.

After 12, 24 and 48 hours at 100°C, approximately 91%, 65% and 35% were measured. After 20 minutes at 110°C, the enzyme activity decreased to 4. 4% and 1 hour later 10% of the enzyme activity. 5 minutes at 120°C Later on, 10% of the enzyme activity was determined. The results are shown in Figure 3.

base 1st person allusion In the presence of 1.3 U/mL pullulanase preparation, 50s+M acetate buffer (pH 6,0) 1% (W/V) substrate was incubated. Incubate Jiji The temperature was 100"C. A 500μL sample was collected and placed in an ion exchanger (Serd olit MB), then HPLC (carbohydrate column Am1nex) HPX-42A).

Pullulan is hydrolyzed in the endo form and has a DP of 3. Forms DP6 and DP9.

After 1 hour, 80% of DP3 was produced. Same as DP3 is maltotriose. DP3 was incubated with α-glucosidase from yeast for the purpose of I turned on. DP3 was completely converted to glucose. Therefore, the pullulanase It is clear that maltotriose was produced by the action. Dextran is this Not affected by enzyme systems.

G2 G2-β-cyclodextrin (Novo Nordisk A/S) 6 maltose was formed in high concentrations.

1.6 - Partially purified pullulanase with the aim of finding the smallest substrate containing a bond Digin was incubated with panose. Interestingly, from known microorganisms Unlike pullulanase, panose was also affected, albeit at a slower rate. This addition The water decomposition products were DPI and DP2. DP2 is the same as maltose by TLC. determined. Therefore, the α-1,6-bond of panose is also hydrated by the enzyme of the present invention. Decomposed.

In addition to these substrates, the pullulanase of the present invention also contains α-amylase and starch ink. It was also possible to attack branched oligosaccharides prepared in the laboratory by oxidation. Next, the shape 80% of the 0-branched oligo scum was separated using the Blo-Ge1 column. The above was broken down into DP5 or less.

Example 4 Cloning of pullulanase of Pyrococcus boisei ・Chromosomal DNA of Poisei, Pitcher et al. (1989).

crobio1. ．． Isolated according to 8, 151-156. and then partially digested with 5au3A. 100μg of P-poisei DNA is Digested with 20 units of 5au3A for 10 minutes at 37°C. The digestive reaction is phenol : Stopped by chloroform extraction, then DNA was ethanol precipitated. Linking The reaction was performed using chromosomal DNA. That is, psJ933 (BamHI (the largest fragment of 5.8 kb was isolated) in a 1:3 ratio of 10μ Using 4 μg of DNA per liter, add 2 units of T4 ligase and incubate at room temperature for 4 hours. I did Cubage Gin. (Plasmid psJ933 was sent to NCIMB for E. coli. It has been deposited as strain 5J989, and the genetic map of the plasmid is shown in Figure 4. Transform E. coli MC100O strain with the ligated DNA, Luria medium supplemented with 2% agar containing 10 μg/a+L of phenicol The cells were inoculated onto the ground and incubated at 37°C. Incubate for 16 hours After that, approximately 14,000 chloramphenicol-resistant colonies were observed on the plate. It was done. These colonies, 2% agar, 6 μg/■L chloramphenicol and replica into fresh 1&l Luria medium containing 0.1% stained pullulan and then The seeds were grown overnight. (Stained pullulan: Pullulan (Hayashiba Bioc) chemical Laboratories) 50g and C1bachros Rot B (Ciba Geigy) 5g and 0.5M NaOH500 mL and incubate the mixture with constant stirring at room temperature for 16 hours. I turned on. The pH was adjusted to 7.0 with 4N H1SO4.

Thereafter, the dyed pullulan was collected by centrifugation, and this pullulan was washed three times with distilled water. It was then suspended in an appropriate amount of distilled water).

Next, these plates were incubated at 60°C for 4 hours, and no colonies appeared. A halo appeared around one of the spots due to decomposition of the dyed pullulan. this first one Isolate the corresponding colonies on a set of Luria medium plates, and add to the plasmid content. I analyzed it. The isolated colony PL2118 was grown in 10 mL of Luria medium. and described in Kieser et al. (Plasmid 12:19, 1984) The plasmid was isolated by the method. This plasmid was analyzed by restriction 72 ping. However, an insert of approximately 4.5 kb of Pyrococcus DNA was found.

Example 5 Dan JiJ The strain of Example 4 was cultured in a 2L Porton type fermenter. (pH was maintained at 6.5-7.0, temperature was 37°C, aeration was 1.ILZ min). and the shaking was at 1000 rpm).

Inoculation was carried out at 37°C on LB agar plates containing 6 g/L chloramphenicol. Reconstitute the night-grown culture into physiological saline solution! %! It got cloudy. Collection is from inoculation to 5 I went at the 0th hour.

The culture was carried out as a batch fermentation using the following medium composition.

Glycerol 60g/L Tryptone (Bacto) 27 g / L Yeast Extract (Bacto) 4 g/LNaCI 1.3 g/L K! 1IPOn 5.3 g/L KHiPOn’ 1.3 g / LLSOa 3.5 g / L CaClt 42Hz0 17mg/LMgSOa ・7HxOO, 67g / LMikrosoy” 20mL/L Brulonifta 1mL/L The pH was adjusted to 7.0 and the medium was autoclaved in a fermenter at 121°C for 40 minutes. Ta.

After sterilization, 15 g glucose and 6 g glucose in 50 L per liter of medium composition. Loramphenicol was added.

”　Mikrosoy　： G/L Ha3citrate 10 Borax (NatBnOt H10HtO) 1.0Mn5Oa ・HtO O, 5 PlISO4・7HzO2,0 CuSOa・5HzOO,2 BaC1g・2)1to0.1 Example 6 11M The culture process prepared according to Example 5 was transferred to 5ervall RC-3B. Centrifuge at 4000rp*/5ervall H6000^ for 30 minutes. I was concerned.

Add the pellet to 5011M Tris buffer (pH 7,0) until the culture reaches 10% of the final volume. It was resuspended so that Add lysozyme to a final concentration of 2 g/L and incubate for an hour, then Ultratorrax/1ype TP1 8/10; 18N; 170W was put into the suspension and treated for 3 minutes.

The suspension was then allowed to dry for 1 hour and centrifuged in a 5-ervall RC-5B centrifuge for 4 hours. Centrifugation was performed at 9000 rpm/5srvall GS3 for 30 minutes at °C.

The pullulanase activity of the supernatant was determined by ultrafiltration through a DDS/GR81PP membrane at room temperature. Concentrated 2.8 times (5mM NaN3 added before ultrafiltration).

Example 7 Deaf-Decision The pullulanase sample prepared according to Example 6 was tested at optimum pH1, optimum temperature, and thermal stability. and characterization of pullulanase activity with respect to substrate specificity.

ball! ! Standing still Pullulanase samples were incubated as follows.

Buffer: 50mM sodium acetate (all 5.0), final concentration.

Substrate = 2% w/v pullulan, panose or soluble starch (Merck) , final concentration.

Temperature: 60℃.

Time: 24 hours.

Stopping the enzymatic reaction: cold at 0°C.

TLC analysis shows that the above pullulanase almost completely converts pullulan into maltotriose. decomposes panose almost completely into glucose and maltose, and converts soluble starch into As hydrolysis products, all glucose oligomers of DP+ and higher It was broken down into things that included. The rate of hydrolysis of pullulan is extremely fast.

Kutama Qualitative A small amount of pullulanase sample sealed in a glass ampoule was heated to 10.30°C at 121°C. or 90 minutes, or 30 minutes at 100°C, 1. 2 or 4 hours in After incubation, it was rapidly cooled. Residual pullulaner at 60°C and pH 5.0 The enzyme activity was measured as described in Example 8.

Blue -ze 0 Up to 1 animal As described on the example day except that various incubation pH/Wk solutions were used. Pullulanase activity was measured at 60°C as described above.

Buffer; pH 3.5-5.5: o, osM sodium acetate (final concentration) = (A buffer ) pH5.5~? , 5: 0.025 M sodium citrate (Has) and 0 ．． 025M HzPOn (final concentration) (CP buffer) incubation p H3, 54, 55, 55, 56, 57, 5 buffer AA AA CP CP C P activity (%) 5 91 100 100 63 3 m! Pullulanase activity in all 5.0 was measured using various incubation temperatures. Measurements were made as described in Example 8, except that

Incubation temperature ("C) 60 70 80 90 100 105 121 Activity (%) 18 38 41 54 98 100 48 Example 8 blue-ze 1 mL of pullulan 4% (w/v) in 0.1 M sodium acetate (pH 5,0) was added to 0.0 The diluted enzyme solution (pullulanase sample was incubated at °C after addition of NazC03 buffer). diluted to yield up to 0.2 g glucose equivalent in the solution mixture IL. added).

Incubate the incubation mixture volume 1a+L for 30 minutes at 60°C. Ta. Incubate 1 mL of incubation mixture for 30 minutes at 0°C. did. Add 1.5 d of 0.5 M NazC island buffer wave buffer 10.0) to each. The mixture was then rapidly cooled.

The amount of reducing sugar in both samples was determined using the Nelson/Sogoi method. (Nelson N, (1944), J. Biol, Chem, 153.3 75-80, and 33υ! 9” (λ14-M, (1952), J, Bio 195.19-23). Bride value (0℃) The activity after subtracting is expressed as follows.

Micromolar glucose equivalent bone x L of culture broth formed (PI/L) Example 9 Saranatsu Kakusei The molecular weight of the pullulanase of the present invention is determined by the first gel gradient (Phast). gel Gradient) 8-25 with Pharmacia Fast System (P harsacia Phast 5yste+w) Follow file No. 110 6 Molecular weight measured by SDS PAGE 95,000 (+/-10,000 ) was measured.

Identification between pullulanase and the M14-95,000 band was determined using 0.1% stained pullulanase. By superimposing the run and gel and incubating at 60”C for 4 hours. went. Only one band with pullulanase activity was detected.

Since the oral HA protein derived from Pyrococcus is only 4.5 kb, The DNA insert does not appear to encode one or more pullulanases.

Activity (%) FIG.1 Activity (%) FIG.2 Activity (%) FIG.3 abstract The present invention is in the field of thermostable enzymes. More specifically, the present invention relates to Pyrococcus (h animals ■) Novel thermostable pullulanase obtained from strains belonging to the genus and methods for producing these enzymes. In addition, the present invention also provides the same type of pullulana The genome of a strain belonging to the genus Pyrococcus consists essentially of Pullulanase produced by recombinant DNA encoding the derived pullulanase Also relates to preparations. Furthermore, the present invention provides a method for using pullulanase components that do not contain contaminating enzyme activity. Concerning high expression production method. Furthermore, the present invention provides starch conversion as well as liquefaction and and/or the use of pullulanase in saccharification processes.

Submission of translation of written amendment (Article 184-8 of the Patent Act) February 1993 (Sun)

Claims (28)

[Claims] 1. Pyrococcus woesei (DSM No. 3773) or Pyrococcus furiosus furiosus) (DSM No. 3638) equivalent to native pullulanase or are characterized by having partially equivalent immunochemical properties. 2. (a) The optimum pH is within the range of pH 5 to 7, (b) The optimum temperature is 85 to H5 (c) incubated in the presence of substrate and calcium. The residual activity, determined later, exceeded 90% after 4 hours at 100°C and 110%. exceeds 30% after 20 minutes at °C, A pullulanase characterized by having the following properties. 3. The protein according to claim 2, which has α-1,6-bond cleavage activity toward panose. Lulanase. 4. A strain belonging to the genus Pyrococcus or a mutant or variant strain thereof The pullulanase according to claim 2 or 3, which can be obtained from . 5. Pyrococcus boisei (DSM No. 3773) or Pyrococcus boisei Claim 4 obtainable from Furiosus (DSM No. 3638) pullulanase. 6. A pullulanase-producing strain belonging to the genus Pyrococcus was used as a carbon and nitrogen source. a step of culturing in a suitable nutrient medium containing mineral salts and inorganic salts; The production of pullulanase according to any one of claims 1 to 5, comprising the step of Construction method. 7. A pullulanase-producing strain belonging to the genus Pyrococcus was prepared without containing elemental sulfur. cultured in a non-turbid nutrient medium and continuously supplemented with N2/CO2 or N2. 7. The method according to claim 6, wherein the culturing is performed while 8. P. woesei or P. furio 7. The method according to claim 6 or 7, wherein a strain of S. sus) is cultured. . 9. P. Boisei (DSM No. 3773) or P. Furiosus (DSM) No. 3638) or mutants or variants thereof are cultivated. The method of Section 8. 10. DN encoding pullulanase derived from a strain belonging to the genus Pyrococcus a pullulanase component consisting essentially of substantially homologous pullulanase components recombinantly produced in A. Lanase preparation. 11. Pyrococcus boisei (DSM No. 3773) or Pyrococcus ・Equivalent to pullulanase derived from Furiosus (DSM No. 3638) or Substantially free from homologous pullulanase components with partially equivalent immunochemical properties. pullulanase preparation. 12. The pullulanase component contains at least 6 U per mg of protein, preferably Ta Claim No. 1, which has a pullulan-degrading activity of at least 20 U per mg of protein. A pullulanase preparation according to either paragraph 10 or paragraph 11. 13. When the apparent molecular weight is 95KD and measured by SDS PAGE, A claim showing a substantially homogeneous band, and said band having pullulan-degrading activity A pullulanase preparation according to any of the ranges 10 to 12. 14. Optimal pH of pullulanase component within the range of 4.5 to 6.0, 85 to 115 Optimum temperature in the range of °C and residual activity of over 60% after 4 hours at 100 °C 14. A pullulanase preparation according to any one of claims 10 to 13. 15. pullulan to maltotriose, panose to glucose and maltose, The claim 1 is capable of decomposing starch into various glucose oligomers having a DP of 1 or more. The pullulanase preparation according to any of paragraphs 10 to 14. 16. The pullulanase component is derived from one species of Pyrococcus, such as P. boisei or The pull according to any of claims 10 to 15, which can be produced by P. furiosus. Lanase preparation. 17. The pullulanase component is P. boisei (DSM No. 3773) or P. Claim 16 Produced by Furiosus (DSM No. 3638) pullulanase preparation. 18. The method for producing high expression of pullulanase according to any one of claims 10 to 17. a step of isolating a DNA fragment encoding pullulanase; The process of combining the protein with appropriate expression signals of a suitable plasmid vector, The lasmid vector can be introduced into a suitable host as an autonomously replicating plasmid or The step of incorporating into the chromoplast, culturing the host microorganism under conditions that express pullulanase. and then recovering pullulanase from the culture. 19. The host microorganism is Escherichia coli or Bacillus spp., Aspergillus spp. 1 belonging to the genus Streptomyces or the genus Streptomyces 19. The method of claim 18, which is a bacterial strain. 20. The plasmid is pBR322 or pACYC or a derivative thereof. and the host microorganism is E. coli. Method. 21. the plasmid is pUB110, pC194 or pE194, and 19. The method of claim 18, wherein the host microorganism is a Bacillus sp. 22. The host microorganisms are B. subtilis and B. licheniform. B. licheniformis, B. amyloliquefaciens (B. ．． amyloliquefaciens) or B. lentu 22. The method of claim 21, wherein s). 23. Boiling the fermentation process causes denaturation of native proteins, and then Remove pullulanase from the supernatant by centrifugation of the fermentation process to remove any contaminating enzyme activity. Claim 10 further comprising a purification step of the expressed pullulanase to recover the the method of. 24. Pullulanase according to any one of claims 1 to 5 in the starch conversion step or the use of a pullulanase preparation according to any of claims 10 to 17. 25. Method of converting starch into glucose and/or maltose containing syrup The pullulanase according to any one of claims 1 to 5 or the pullulanase according to claim 1 The pullulanase preparation according to any of items 10 to 17 and glucoamylase, α-glucan one or more enzymes selected from cosidase, β-amylase or other saccharifying enzymes; A method comprising the step of saccharifying a starch hydrolyzate in the presence of. 26. 1 g of dry matter in the temperature range of 60 to 80 °C with a pH range of 4.0 to 6.0 for saccharification The starch conversion according to claim 25 carried out at a dose of 1 to 100 μg of pullulanase per method. 27. Method of converting starch into glucose and/or maltose containing syrup The pullulanase according to any one of claims 1 to 5 or the pullulanase according to claim 1 Simultaneous liquefaction/debranching in the presence of a pullulanase preparation according to any of paragraphs 10 to 17. and then glucoamylase, α-glucosidase, β-amylase or One or more enzymes selected from other saccharifying enzymes (in some cases, pullulanase is also included) 2) A method comprising the step of performing saccharification in the presence of 28. Simultaneous liquefaction/debranching to a starch slurry of 25-45% by weight on dry matter. On the other hand, in the temperature range of 95 to 130 °C with a pH range of 4.0 to 6.0, pullulanase dose of 1 to 100 μg per gram of dry matter and thermostability α of 1 to 20 μg per gram of dry matter. - A process for converting starch according to claim 27, carried out at amylase doses.