Neutral low-temperature xylosidase CaXyl43A and gene and application thereof

A technology of xylosidase and low temperature, which is applied in the field of genetic engineering and can solve problems such as loss of enzyme activity

Active Publication Date: 2016-08-17
INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Most xylosidases of filamentous fungal origin are mesophilic or hypertherm

Method used

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  • Neutral low-temperature xylosidase CaXyl43A and gene and application thereof
  • Neutral low-temperature xylosidase CaXyl43A and gene and application thereof
  • Neutral low-temperature xylosidase CaXyl43A and gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 Enzyme-producing characteristics of Cladosporium acalyphae SL-16

[0044] After culturing Cladosporium acalyphae SL-16 in potato juice medium, the spore suspension was made into bran liquid medium (Luo et al. Enzyme Microbial Technology, 2009, 45:126–133), 15 Cultivate at ℃ for 7 days, use 1mM p-nitrophenyl-β-D-xylose as substrate, react at pH 6.0 and 30℃ for 10min, and determine that it has xylosidase activity by spectrophotometer.

Embodiment 2

[0045] Example 2 Cladosporium acalyphae SL-16 Cloning of Xylosidase Encoding Gene Caxyl43A

[0046] Extraction of Cladosporium acalyphae SL-16 genomic DNA:

[0047] Filter the mycelium cultured in liquid for 3 days with sterile filter paper, put it into a mortar, add 2mL of extract, grind for 5min, then put the grinding solution in a 50mL centrifuge tube, lyse in a water bath at 65°C for 20min, and mix every 10min. Homogenize once and centrifuge at 10,000 rpm for 5 min at 4°C. The supernatant was extracted in phenol / chloroform to remove impurity proteins, and then an equal volume of isopropanol was added to the supernatant. After standing at room temperature for 5 minutes, centrifuge at 10,000 rpm for 10 minutes at 4°C. The supernatant was discarded, the precipitate was washed twice with 70% ethanol, dried in vacuum, dissolved by adding an appropriate amount of TE, and stored at -20°C for later use.

[0048] Design and synthesize specific primers Caxyl43A-F / Caxyl43A-F:

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Embodiment 3

[0052] Example 3 RT-PCR Analysis of Cladosporium acalyphae SL-16 Xylosidase Gene

[0053] Extract the total RNA of Cladosporium acalyphae SL-16, use reverse transcriptase to obtain a strand of cDNA, and then amplify the single-stranded cDNA with primers Caxyl43A-E-F / Caxyl43A-E-F to obtain the cDNA sequence of xylanase, After the amplified product was recovered, it was sent to Sanbo Biotechnology Co., Ltd. for sequencing.

[0054] Caxyl43A-E-F: 5'-atgaccgaccctacacagaaagacc-3';

[0055] Caxyl43A-E-R: 5'-ggggaattcctagtggtggtggtggtggtgctcctgcggctgcgccaacttg-3';

[0056] By comparing the genome sequence and cDNA sequence of xylosidase, it is found that the gene has no intron, encodes 329 amino acids and a stop codon, and has no signal peptide sequence. The isolated and cloned gene encoding xylosidase is a new gene.

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Abstract

The invention relates to the field of gene engineering, in particular to neutral low-temperature xylosidase CaXyl43A and a gene and application thereof. The amino acid sequence of the xylosidase is shown in SEQ ID NO.1. The xylosidase has the properties including the optimal pH of 6.8, the optimal temperature of 35 DEG C, the enzyme activity maintenance property at 0 DEG C, and the specific activity of 181.6 U/mg, has the activities of xylosidase, Arabinfuranosidease and circumscribed xylanase, and has certain xylose tolerance and the capacity of completely degrading xylo-oligosaccharide to generate xylose. Industrial fermentation production is easy. As a novel broad-spectrum enzyme preparation, the xylosidase can be widely used for aquatic feed industry, food industry, papermaking industry, energy industry and the like.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular, the invention relates to a neutral low-temperature xylosidase CaXyl43A and its gene and application. Background technique [0002] Xylan (xylan) is the main component of plant biomass, and its content in nature is second only to cellulose (Lynd et al. Microbiology and Molecular Biology Review, 2002, 66:506–577), and its effective utilization is of great significance . The biodegradation of xylan requires xylanase (EC 3.2.1.8) to cleave polysaccharides into xylooligosaccharides, which are further degraded into xylose by xylosidase (EC 3.2.1.37) (Collins et al. al. FEMS Microbiology Review 2005, 29:3–23). Therefore, xylosidase acts as a rate-limiting enzyme in the process of xylan degradation, which can alleviate the product inhibition of xylanase (De Almeida et al. FEMS Microbiology Letters 1995, 130: 171-175). A variety of microorganisms, including bacteria, archaea, fungi, et...

Claims

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Application Information

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IPC IPC(8): C12N9/42C12N15/56C12N15/63
Inventor 姚斌马锐柏映国黄火清罗会颖苏小运王亚茹王苑师霞
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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