TH31111B - Recombinant, a heat resistant enzyme that releases tetrahalose from non-reducing saccharides. - Google Patents

Abstract

It revealed recombinant thermostable enzymes with a molecular weight of approximately 54,000 to 64,000 coltons and a PI of approximately 5.6-6.6 and released trehalose from nonreducing. Saccharides with a trehalose structure are end units. and there is de-glucose polymerization of At least 3 enzymes have satisfactory high thermal stability. that is, it can be held that it was not inactivated even after incubation in acaus solution (pH 7.0) at 85 °C for 60 min, and this complemented the industrial scale production of trehalose. and in a satisfactory high yield

Claims (8)

1. Recombinant, heat-resistant enzyme with the following physicochemical properties: (1) Action-releasing tri-haloose from non-redoxaccharide with a tree structure. Halos as The endocrine and at least 3 (2) degrees of glucose polymerization have a molecular weight of approximately 54,000 to 64,000 coltons on sodium codecyl sulfate polyacrylic. Lamide Gel Electrophoresis (SDS-PAGE) (3), isoelectric point (pI) approx 5.6 to 6.6 on isoelectrophoresis, and (4). Stability to temperature Not significantly inactivated Even incubated in aqueous solution (pH7.0) at 85 ํ for 60 min. 2. The heat-resistant enzyme recombinant referred to in claim 1 with the amino acid sequence. Amino selected from a group containing an amino acid sequence from the N end in the SEQ ID NO: 1 and the homologous amino acid sequence against it. 3. The recombinant dNA code. Heat-resistant enzyme As claimed in claim 1 4. The DNA claimed in claim 3 contains a sequence of selections from the group containing the beam. Extinguish the bass from the end of 5 \ 'in SEQ ID NO: 2, the bass sequence at Hom Alogus against it and the sequence at 5. DNA claimed in claim 3, where one or more bases are replaced in the SEQ ID NO: 2 sequence with another base by the code generator. Genetically without replacement The amino acid sequence in SEQ ID NO: 1 6. The DNA claimed in claim 3 that the life-giving derivative of the family Sulfolobus 7. Self-replicable DNA with a capable vector Can simulate itself and the DNA that Code of recombinant heat-resistant enzyme of claim 1 8. Self-replicated DNA recombinant as claimed in claim 7 with a quench value. Selected from a group consisting of a baseline from tip 5 \ 'in SEQ ID NO: 2, a homologous sequence against it, and a complementary base sequence to these sequences. 9. Recombinant. Self-replicating DNA as claimed in claim 7, where replacing one base is greater than in the SEQ ID NO: 2 sequence with another base by the DJ's Genetic code without amino acid sequencing in SEQID NO: 1 1 0. Self-replicated DNA recombinant with self-replicating vector and coded DNA. Of recombinant, heat-resistant enzyme of claim 1, where the DNA derivative of the genus Sulfolobus 1 1. A self-replicated recombinant DNA as required by Claimed in Claim 7 or Claim 10, where such a self-replicating vector is Plasmidwector BlueCrypt II SK (+) 1 2. Trans A formant that can be obtained by inserting a self-replicating recombinant DNA containing a vector The heat-resistant enzyme recombinant of claim 1 is entered into the appropriate host. 1 3. Transformant as claimed. The right is placed in claim 12, where the DNA has a selected base sequence from that group. Contains the base sequence from the tip 5 \ 'in the SEQ ID NO: 2 homologous base sequence against it and These complementary sequences 1 4. Transformant, as claimed in claim 12, where such DNA is prepared with one base substitution. Or greater in the SEQ ID NO: 2 sequence with another base by the gener C of the genetic code without change. Amino Acid Sequence SEQ ID NO: 1 1 5. Transformant as claimed in claim 12, where the DNA is derivative. From the microorganisms of the genus Sulfolobus 1 6. Transformant, as claimed in claim 12, where self-replicable vectors can be found. Such is the Plasmic Vector Blue Script II SK (+) 1 7. Transformant as claimed in claim 12, where the said host is. Escherichia coli species 1 8. Procedures for preparing heat-resistant enzyme recombinants consisting of culture. A transformant prepared by inserting a self-replicating recombinant DNA containing a vector that Self-replicating and DNA code of recombinant, heat-resistant enzyme of Claim No. 1 goes into the appropriate host. And store recombinant anzyme that is resistant to the heat that formed it From cell only 1. 9. Method, as claimed in claim 18, where the aforementioned DNA has a sequence selected from the group that is Contains the base sequence from tip 5 \ 'in the SEQ ID NO: 2 sequence where the Homologas against it and The complementary sequence of these bases 2 0. The method claimed in Proposition 18 where such DNA is prepared with the base substitution. One or more characters in the SEQ ID NO: 2 sequence with another base based on the genesis of the genetic code. Without changing the amino acid sequence in SEQ ID NO: 1 2 1. Process referred to in claim 18, where the DNA is a microbial derivative of the Sulfolobus family 2. 2. The process referred to in claim 18, where such self-replicating vector is Plast Midwector Blue Script II SK (+) 2 3.Procedures, as claimed in claim 18, where the host is the Escherichia coli 2 species microbe. 4. The process referred to in claim 18, where the heat-resistant enzyme recombinant is stored. Obtained by one or more techniques selected from the group containing Centrifugation, filtration, concentration Salt removal, dialysis, separation by exposure, ion exchange chromatography. Gel Filtration Chromatography Hydrophobid chromatography chromatography Gel Electrophoresis And iso electrophoresis 2 5.Method for enzymatic conversion with a step that allows recombinant enzyme The resistance of claim 1, active on non-redoxaccharide with a trihalose structure, is The end units and the degree of glucose polymerization are at least 3 to release the trihalose 2. 6. Method as claimed in claim 25 with the release of the recombinant procedure. The heat-resistant enzyme is co-contained in aqueous solution containing 50 w / w% or less of the non-redoxaccharide based on dry solid weight and allowed to recom. Binant enzyme That is resistant to such heat effects on sleep - Reducing saccharides above 55 ํ 2 7. Method as claimed in claim 25, where non-reducing saccharide was obtained. It was activated by enzymes that form non-redoxaccharides and non-redoxinization. The obtained saccharides through the inactivation of recombinant, heat-resistant enzymes 2. 8. The method referred to in claim 25, where non-reducing saccharide is Members selected from the group that consisted of 4-Glucosyl Terehaloos, 4-Maltosil Terehalos, 4-Malto Triosiltrihalos, 4-Malto Tetrao Sil-Trehalose, 4-Malto Penta, Cyl Trehalose and its mixtures.