Method for detecting activity of beta-1,4-xylosyltransferase in xylan synthesis by utilizing high performance liquid chromatography (HPLC)

A technology for xylose transferase and xylan, which is applied to measurement devices, instruments, scientific instruments, etc., can solve the problems of difficult operation and cumbersome methods, and achieve the effects of simple operation, high sensitivity and good reproducibility.

Inactive Publication Date: 2014-07-30
SOUTH CHINA AGRI UNIV
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  • Description
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Problems solved by technology

Although it has been reported that the addition reaction of XylT can be determined by isotope analysis, the method is cumbersome, requires strict experimental conditions, and is not easy to operate

Method used

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  • Method for detecting activity of beta-1,4-xylosyltransferase in xylan synthesis by utilizing high performance liquid chromatography (HPLC)
  • Method for detecting activity of beta-1,4-xylosyltransferase in xylan synthesis by utilizing high performance liquid chromatography (HPLC)
  • Method for detecting activity of beta-1,4-xylosyltransferase in xylan synthesis by utilizing high performance liquid chromatography (HPLC)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Embodiment 1 Xylo-oligosaccharides containing fluorescent groups (Xyl n -AA) mark

[0056] ① Preparation of derivatization reaction reagents: 30mg / mL fluorescent group 2-aminobenzoic acid (Anthranilic Acid, AA) and 20mg / mL sodium cyanoborohydride (NaBH 3 CN) was dissolved in MABS to obtain the derivative reaction

[0057] Reagents; Among them, MABS (Methanol, Acetate, Borate mixed solution)

[0058] For: 24mg / mL sodium acetate and 20mg / mL boric acid are dissolved in methanol solution, and MABS should be prepared and used immediately;

[0059] ② Labeling reaction: xylooligosaccharide (Xyl n , n is 1 to 6) (purchased from the Carbohydrate Research Center of the University of Georgia, USA, http: / / www.ccrc.uga.edu / services / index.php) and the derivatization reaction reagent obtained in step ① according to the mass volume ratio of 1mg : Mix 1mL, react at 80°C for 80min, add diethyl ether (diethyl ether, analytically pure) to mix the excess derivatizing reagent and extract...

Embodiment 2

[0061] Embodiment 2 establishes HPLC to detect Xyl n -AA method

[0062] (1) Preparation of Xyl 1 -AA~Xyl 6 - Individual standard samples and mixed standard samples of AA, each with a final concentration of 0.1 mM.

[0063] (2) Determine the peak elution time of each standard sample and mixed standard samples by HPLC. The chromatographic conditions are: chromatographic column ZORBAX Eclipse XDB-C18 column (2.1mm×250mm, 1.8μm) (Agilent, purchased from Agilent Technologies Co., Ltd.), mobile phase: A phase is 50mM sodium acetate aqueous solution (pH4.3), B phase Chromatographically pure acetonitrile (Honeywell, 99.99% pure, purchased from Shanghai Dingguo Biotechnology Co., Ltd.), flow rate: 0.5mL / min, column temperature: 20°C, detector: Agilent1100HPLC systems (purchased from Agilent Technologies Ltd.), Ex 320nm ,Em 420nm , injection volume 5μL, recording time: 42min. Gradient elution program: 0~5min (8% phase B), 5~25min (20% phase B), 25~30min (40% phase B), 30~35min (...

Embodiment 3

[0066] Example 3 Analysis of XylT activity of japonica

[0067] 1. Extraction of yellow beam wood microcapsules

[0068] Take the peeled stem segments of the upper, middle and lower parts of the annual Neolamarckia cadamba (Rubiaceae) (collected in Tianhe Park, Guangzhou), and add them to the extract solution (100mM Hepes-KOHbuffer pH6.8, 1mM DTT, 1mM EDTA , 0.1 mM MnCl 2 , 0.4M sucrose; Among them, the Hepes-KOH buffer of 100mM pH6.8 is the solution of 23.831g 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) dissolved in deionized water, and the volume is adjusted to 1L, and the 100mM Hepes solution is adjusted with KOH pH to 6.8. ) was homogenized in a high-speed tissue grinder (model DS-1, purchased from Shanghai Specimen Model Factory) at 4°C (the weight-to-volume ratio of the plant to the extract was 2g: 1mL), and filtered through a filter cloth (cat.475855, Miracloth, Calbiochem Company) for filtration, the obtained filtrate was centrifuged at 10,000g, 4°C for 10mi...

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Abstract

The invention discloses a method for detecting the activity of beta-1,4-xylosyltransferase in xylan synthesis by utilizing high performance liquid chromatography (HPLC). The method comprises the following steps: extracting plant microcapsules, carrying out an addition reaction between Xyln-AA and UDP-Xyl by utilizing beta-1,4-xylosyltransferase existing in the microcapsules, detecting the addition product by utilizing the HPLC, and detecting the activity of XylT in plants. Because XylT is a key enzyme in xylan synthesis, and the xylan is a main ingredient of biomass hemicellulose, the biomass hemicellulose is well utilized by understanding a xylan synthesis mechanism. The invention provides a reliable way for deeply researching the xylan synthesis mechanism and regulating xylan ingredient in hemicellulose, genes formed by xylan can be researched and regulated by screening xylan synthesis defect plants according to the needs, or the requirements on different contents of the hemicellulose in materials are met by regulating key genes influencing XylT synthesis.

Description

technical field [0001] The invention belongs to the field of biological detection, in particular to a method for detecting the activity of β-1,4-xylosyltransferase (Xylosyltransferase, XylT) in xylan synthesis by using HPLC. Background technique [0002] Cellulose, hemicellulose and lignin are the main components of agricultural and forestry biomass resources, and understanding their synthesis process in plants is conducive to better utilization of these biomass resources. Xylan is the main hemicellulose of the secondary wall of monocot and dicot plants, the main chain is D-xylan-xylan linked by β-1,4-glycosidic bonds, and XylT is the catalytic uridine diphosphate xylose (UDP-Xylose, UDP-Xyl) is a key enzyme added to the xylan backbone, located in the inner membrane, and can promote the elongation of the xylan backbone. [0003] The research on the xylosyltransferase (XylT) gene involved in the extension of the xylan backbone is relatively late, and the first article was pu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02
Inventor 吴蔼民赵先海邓小梅陈晓阳宋丽丽
Owner SOUTH CHINA AGRI UNIV
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