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Neutral cold-adaptive xylanase CaXyn11A as well as gene and application thereof

A technology of xylanase and low temperature, applied in the field of genetic engineering, can solve the problem of loss of enzyme activity

Active Publication Date: 2016-07-27
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 xylanases of filamentous fungal origin are mesophilic or hyperthermic enzymes that lose enzymatic activity at ambient or cryogenic conditions

Method used

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  • Neutral cold-adaptive xylanase CaXyn11A as well as gene and application thereof
  • Neutral cold-adaptive xylanase CaXyn11A as well as gene and application thereof
  • Neutral cold-adaptive xylanase CaXyn11A as well as gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Embodiment 1 Cladosporium acalyphae SL-16 produces enzyme characteristic

[0048] After culturing Cladosporium acalyphae SL-16 in potato juice medium, the spore suspension was prepared and inoculated in bran liquid medium (Luoetal. % beech xylan was used as the substrate, reacted at pH 6.0 and 30°C for 10 min, and determined to have xylanase activity by the DNS method (Miller1959).

Embodiment 2

[0049] Cloning of Example 2 Cladosporium acalyphae SL-16 Xylanase Encoding Gene Caxyn11A

[0050] Extraction of Cladosporium acalyphae SL-16 genomic DNA:

[0051] 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.

[0052] Design and synthesis of specific primers Caxyn11A-F / Caxyn11A-F:

[0...

Embodiment 3

[0056] RT-PCR Analysis of Example 3 Cladosporium acalyphae SL-16 Xylanase Gene

[0057] Extract the total RNA of Cladosporium acalyphae SL-16, use reverse transcriptase to obtain a strand of cDNA, then amplify the single-stranded cDNA with primers Caxyn11A-E-F / Caxyn11A-E-F, obtain the cDNA sequence of mature xylanase, and amplify The recovered product was sent to Sanbo Biotechnology Co., Ltd. for sequencing.

[0058] Caxyn11A-E-F: 5'-GGGCATATGATGGTCGGAATCAAGTCCCTTCTCCTCGC-3';

[0059] Caxyn11A-E-R: 5'-GGGGAATTCCTAGTGGTGGTGGTGGTGGTGAGACGTCTG

[0060] AACGTAGATGTCAGCG-3'.

[0061] By comparing the genome sequence and cDNA sequence of xylanase, it was found that the gene had 2 introns, the cDNA was 696 bp long, encoded 231 amino acids and a stop codon, and the N-terminal 19 amino acids were its signal peptide sequence. The measured partial nucleotide sequence of the mature protein of the gene Caxyn11A was homologously compared with the xylanase gene sequence on GenBank, and th...

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Abstract

The invention relates to the field of genetic engineering, in particular to neutral cold-adaptive xylanase CaXyn11A as well as a gene and an application thereof. The amino acid sequence of the neutral cold-adaptive xylanase CaXyn11A is as shown in SEQ ID NO.1; the xylanase disclosed by the invention has the following properties: optimum pH value is 6.5, the optimum temperature is 40 DEG C, an enzyme activity is about 50% at 20 DEG C and a specific activity is 357.3U / mg, a catalysis efficiency at ambient temperature (20 DEG C) is higher than the optimum temperature (40 DEG C), and the xylanase is easy for industrial fermentation production. The xylanase, as a novel enzyme preparation, can be widely applied to aquatic feed, food, paper making, 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 xylanase CaXyn11A and its gene and application. Background technique [0002] Xylan (xylan) is the main component of plant cell wall hemicellulose, its content in nature (20-35%) is second only to cellulose (35-50%), and provides a rich material basis for bioenergy. Compared with the high-efficiency utilization of plant cellulose, xylan is difficult to effectively utilize, and it is directly discharged into the water body as industrial waste, which leads to the enrichment of water quality nutrients and seriously damages the ecological environment. Xylanase is a general term for a class of enzymes that degrade xylan to generate oligosaccharides and xylose, most of which are classified in the 10th, 11th and 43rd families of glycoside hydrolases. The xylanase of the 11th family (EC3.2.1.8) is called a true xylanase because it speci...

Claims

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Application Information

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IPC IPC(8): C12N9/42C12N15/56C12N15/63
CPCC12N9/2482C12Y302/01008
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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