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Thermophilic chaetomium cellulase, as well as preparation method and application thereof

A thermophilic chaetomium cellulase technology, applied in the field of cellulase, thermophilic Chaetomium cellulase, thermophilic Chaetomium cellulase, can solve the unsatisfactory effect of acid cellulase and other problems, to achieve obvious effects and application effects

Active Publication Date: 2013-10-09
希杰尤特尔(湖南)生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the polishing process of this kind of fiber mostly adopts more dosage and short-time polishing, but the effect of using acid cellulase is not ideal at present
At present, there are no related reports about using Trichoderma reesei or Pichia pastoris to express cellulase from Chaetomium thermophila

Method used

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  • Thermophilic chaetomium cellulase, as well as preparation method and application thereof
  • Thermophilic chaetomium cellulase, as well as preparation method and application thereof
  • Thermophilic chaetomium cellulase, as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] 1. Microorganisms and plasmid materials

[0051] The Escherichia coli DH5α, Pichia pastoris and pPink-HC plasmids used for cloning were provided by Invitrogen. The host used for the high-efficiency expression of heterologous genes is Trichoderma reesei RUT C-30, purchased from American Type Culture Collection (ATCC), and the starting plasmid pBluescipt KS (1) was purchased from Stratagene.

[0052] 2. Discovery and Synthesis of Cellulase Gene

[0053] Using Trichoderma reesei endo-β-1,4-β-glucase V (EGV, code number: CAA83846; Saloheimo et al., 1994. Mol. Microbiol. 13: 219-229) as a template, search with NCB1 / BLAST Homologous protein series, found that Chaetomicum thermophilum (Chaetomicum thermophilum) genome library has a homologous protein with unknown function. The gene sequence, cDNA sequence and protein sequence are as shown in SEQ ID NO1, SEQ ID NO2 and SEQ ID NO3, respectively. Show. In order to express efficiently in Trichoderma reesei, Life Technology Corporati...

Embodiment 2

[0059] Example 2 Test of the effect of CHTCA cellulase produced by Trichoderma reesei on the hair removal of textiles

[0060] Put the same amount (0.03g) of CHTCA cellulase (prepared in Example 1) and commercial cellulase (all-round enzyme 11L purchased from Genencor) and 25×25cm white knitted fabric under 10 liters of various pH conditions Process for 40 minutes. The speed of the washing machine is controlled at 150RPM and the temperature is 50°C. After washing, rinse the white cloth with clean water for 1 minute. Before and after washing, the white cloth is dried in an oven at 80°C to a constant weight. The polishing effect of removing fluff is as follows Figure 3A , Figure 3B with Figure 3C Shown: Under the same protein dosage, CHTCA cellulase can remove fluff and achieve polishing effect better than commercial cellulase. Moreover, the amount of CHTCA cellulase to remove fluff from pH 4.5-7.5 is more than that of commercial cellulase (see Table 2).

[0061] Table 2. The ef...

Embodiment 3

[0063] Example 3 High-efficiency expression of CHTCA catalytic domain and secretion of recombinant protein in Pichia pastoris

[0064] A DNA molecule sequence (SEQ ID NO. 4) was synthesized by Life Technology, which encodes the 22 to 238 amino acids (SEQ ID NO. 5) of the CHTCA protein corresponding to the CHTCA catalytic domain. A TAA was added to the 3'end of SEQ ID NO. 4 for translation termination. At the same time, for the convenience of cloning, Stu I and Kpn I series were added to the 5'and 3'ends of SEQ ID NO. 4, respectively, SEQ ID NO. 4 After the molecule was digested by these two enzymes, it was ligated with the pPink-HC plasmid after the same enzyme digestion with T4 ligase, and the ligation reaction solution was used to transform E. coli DH5α cells. Eight colonies on LB / Amp agar were selected and placed on LB After overnight culture in liquid medium, the plasmid was isolated, and after DNA sequencing, the correctly constructed plasmid was obtained, named batch pCHT45...

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Abstract

The invention discloses a thermophilic chaetomium cellulase the amino acid sequence of which is selected from the following sequences: a) N-terminal amino acid with the sequence as shown in SEQ ID No.5; b) C-terminal amino acid with the sequence as shown in SEQ ID No.3 or SEQ ID No.5; c) an amino acid sequence containing the sequence formed by replacing, deleting, inserting or adding one or more amino acid residues in the sequence of (a) or (b) and having cellulase activity together with (a) or (b); d) an amino acid sequence containing the sequence having at least 90% of homology with protein of the sequence as shown in SEQ ID No.3 or SEQ ID No.5 and having the cellulase activity. In addition, the invention also discloses a preparation method of the thermophilic chaetomium cellulase expressed by trichoderma reesei and pichia pastoris. The invention further discloses an application of the thermophilic chaetomium cellulase in preparing hair-removing products for textile fabrics and washing products for jeans.

Description

Technical field [0001] The present invention relates to the field of biological technology, in particular to a cellulase, in particular to a Chaetomium thermophila cellulase; in addition, the present invention also relates to a preparation method and application of the Chaetomium thermophila cellulase. Background technique [0002] Cellulase is a high-efficiency catalyst for the hydrolysis of cellulosic substances, which can degrade cellulose into small molecular cellulose, polysaccharide compounds and glucose. Cellulase has existed in some herbivores for a long time. Scientists have studied cellulase for more than 100 years. At the beginning, they mainly wanted to use cellulase to degrade the most abundant cellulose on the earth and turn it into usable bio-energy. In this process, people have discovered many other uses of cellulase. Modern cellulase is widely used in textile printing and dyeing, papermaking, feed, bio-energy, food processing, brewing, environmental protection, o...

Claims

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Application Information

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IPC IPC(8): C12N9/42C12N15/56C12N15/80C12N15/81D06M16/00D06L1/12
Inventor 李新良张永金路代国
Owner 希杰尤特尔(湖南)生物科技有限公司
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