Endo-1,3-β-glucanase, polynucleotide, recombinant vector, transformant, method for producing endo-1,3-β-glucanase, enzyme preparation, and low-molecular paramylon manufacturing method
A technology of molecularized euglena and dextranase, which is applied in the field of manufacturing low-molecular paramylon, can solve the problems of insufficient popularization and poor price competitiveness, etc.
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Embodiment 1
[0138] (Example 1: Preparation of β-1,3-glucanase from Euglena)
[0139] Euglena gracilis (E. gracilis) Z strain was suspended in phosphate buffer (10 mM, pH 7.0), and then crushed by an ultrasonic breaker to obtain a crushed Euglena gracilis solution. After centrifuging (22000xg, 15 minutes) the euglena crushed solution, the supernatant was recovered, and ammonium sulfate (420 g / L) was added thereto.
[0140] This solution was left to stand at 4° C. for 30 minutes, and then centrifuged (22000×g, 15 minutes) to obtain a precipitate.
[0141] The precipitate was dissolved in a phosphate buffer (10 mM, pH 7.0) containing 0.2 M ammonium sulfate, and applied to a hydrophobic column (HiPrep phenyl, GE Healthcare) buffered with the same buffer.
[0142] After the column was washed with a phosphate buffer (10 mM, pH 7.0) containing 0.2 M ammonium sulfate, the concentration of ammonium sulfate was decreased to elute the protein bound to the column.
[0143] figure 1 In , the lamina...
Embodiment 2
[0148] (Example 2: Determination of the amino acid sequence of β-1,3-glucanase derived from Euglena)
[0149] The protein in Example 1 was partially purified, and the active parts of the component numbers 26 to 36 that showed decomposing activity to laminarin in the ion exchange column were used for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS- PAGE) was separated on a 12.5% gel, and stained with Coomassie Brilliant Blue R-250 (CBB, Coomassie Brilliant Blue). Figure 4 In , the fluorescent image of the gel obtained by electrophoresis is shown. Such as Figure 4 As shown, four protein bands were detected.
[0150] The stained protein was excised from the gel as a gel slice and digested with trypsin to obtain a fragmented peptide mixture.
[0151] The trypsinized fragmented peptide mixture was prepared according to the method of Kawamura et al. (Kawamura, Y., and Uemura, M. (2003) Mass spectrometric approach for identifying putative plasma membrane protein...
Embodiment 3
[0155] (Example 3: Identification of polynucleotides encoding β-1,3-glucanase EgCel17A, EgCel81A, EgCel81B derived from Euglena)
[0156] Euglena gracilis (E. gracilis) Z strain was cultured at 29° C. for 10 days in 10 L of Koren-Hutner medium prepared based on the description of “Euglena Physiology and Biochemistry” (Edited by Shozaburo Kitaoka, KKK Publishing Center). Concentrate to about 6 times by centrifugation, and nitrogen gas is passed through the medium until the dissolved oxygen concentration is 0.01 mg / L. What was obtained by capping tightly and leaving still for 24 hours was used as anaerobically treated cells.
[0157] Total RNA was extracted from Euglena (100 mg) recovered by centrifugation using an RNA extraction kit (Qiagen). cDNA was synthesized from the prepared total RNA using oligo(dT) primers and reverse transcriptase (Transcriptase III, Invitrogen). From the gene sequences of EgCel17A, EgCel81A, and EgCel81B parts of amino acid sequences 2, 4, and 6 obt...
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