Novel externally tangent-ª‰-1,4-glucanase/internally tangent-ª‰-1,4-xylanase and application thereof
A technology for the activity of glucanase and xylanase, which is applied in the field of producing simple sugars and glucose, and can solve problems such as inability to adapt
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Embodiment 1
[0067] Purification of apple snail exo-β-1,4-glucanase / endo-β-1,4-xylanase:
[0068] Apple snails are widely distributed in Fujian, Guangdong, Guangxi, Zhejiang, Jiangsu and other provinces in China. Apple snails were introduced from South America 20 years ago as food. They like to eat seedlings. Even if the water quality is poor, apple snails can multiply and grow, becoming rivers. and a scourge in rice fields. The apple snails purchased in the market were used as experimental materials.
[0069] (1) Extract gastric juice from apple snails and precipitate with 0.5 saturation degree ammonium sulfate.
[0070] (2) Collect the precipitate, dissolve it with 10mM phosphate buffer (containing 0.1M sodium chloride and 1mM EDTA), and dialyze.
[0071] (3) Purify by column chromatography on DEAE-Sephadex A-50, and collect the passing peaks.
[0072] (4) Purified by Bio-gel P-100 column chromatography, and collected the hydrolysis activity peak of pNPC (p-Nitrophenyl β-D-cellobiosid...
Embodiment 2
[0076] Amino acid sequence determination of exo-β-1,4-glucanase / endo-β-1,4-xylanase
[0077] 2.1 Main reagents and instruments
[0078] PVDF membranes were purchased from Millipore Corporation. Endo-Glu C protease was purchased from Promega. DTT was purchased from Boehringer Corporation. Iodoacetic acid (ICH 2 COOH) was purchased from Sigma Company. The electrotransfer system uses 2117-250 NOVABLOT Electrophoretic Transfer Kit from LKB Company.
[0079] 2.2 Exo-β-1,4-glucanase / endo-β-1,4-xylanase native N-terminal amino acid sequence determination
[0080] Exo-β-1,4-glucanase / endo-β-1,4-xylanase pure enzyme preparation is loaded for SDS-PAGE, in which the stacking gel is 5%, the separating gel is 10%, each well 70ug of sample was loaded, the electrophoresis voltage was 70V for 30 minutes, and then 125V for 90 minutes.
[0081] After electrophoresis, the separation gel was transferred to the membrane by semi-dry method. After transfer, stain the PVDF membrane with 0.2% ...
Embodiment 3
[0088] cDNA cloning of exo-β-1,4-glucanase / endo-β-1,4-xylanase
[0089] 3.1 Instrument
[0090] The PCR instrument selects SingleBlock from Ericomp Company of the United States TM system.
[0091] 3.2 RNA extraction and reverse transcription
[0092] The gastric tissue of fresh apple snails was extracted with Trizol to extract total RNA, and the total RNA was extracted with oligo(dT) 18 The primers were used for reverse transcription to generate cDNA as a template for PCR.
[0093] 3.3 The first step of PCR
[0094] According to the two amino acid sequences obtained by protein sequencing, the following PCR degenerate primers were synthesized:
[0095] Primer 1A:
[0096] 5′-GCA(T / G / C) GCA(T / G / C) GGA(T / G / C) GCA(T / G / C) GGA(T / G / C)
[0097] GTA(T / G / C) AC-3' (SEQ ID NO: 5)
[0098] Primer 1B:
[0099] 5'-GG A(G)TC A(T / G / C)GC A(T / G / C)GC A(G)TG A(T / G / C)GCA(G)AT-3'(SEQ ID NO: 6)
[0100] Using the cDNA obtained by reverse transcription as a template, the DNA amplification ...
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