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Beta-glucosidase Cel1b, expression gene and application thereof

A glucosidase and gene technology, which is applied to beta-glucosidase Cel1b and its expression genes and application fields, can solve the problems of relatively limited application conditions and few types of beta-glucosidase, and achieves broad application prospects. Effect

Inactive Publication Date: 2012-06-13
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, there are few types of β-glucosidases with clear transglycosylation, and the application conditions are relatively limited.

Method used

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  • Beta-glucosidase Cel1b, expression gene and application thereof
  • Beta-glucosidase Cel1b, expression gene and application thereof
  • Beta-glucosidase Cel1b, expression gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1 Gene isolation, site-directed mutagenesis, heterologous expression and purification of β-glucosidase Cel1b

[0023] (1) Inoculate Trichoderma reesei spores 10 in Trichoderma reesei MM medium 6 Cultured at 30°C for 2 to 3 days, collect hyphae, extract RNA, and reverse transcription to obtain cDNA;

[0024] Composition of Trichoderma reesei MM medium:

[0025] Peptone, 2g; Glucose, 20g; (NH 4 ) 2 8O 4 , 5g; K 2 HPO 4 , 15g use 1M NaOH to adjust the pH to 5.5, after constant volume value of 1L, dispense 200ml per bottle, add 200×MgSO to each bottle after sterilization 4 (120g / l)1ml; 200×CaCl 2 (120g / l)1ml; 100×uridine (1M), 2ml; 1000×trace element (FeSO 4 ·7H 2 O, 5g / l; MnSO 4 ·H 2 O, 1.6g / l; ZnSO 4 , 1.4g / l; CoCl 2 , 2g / l), 200μl.

[0026] The RNA was extracted using the Trizol method, and the Trizol reagent was purchased from Invitrogen. For the steps, refer to the product manual.

[0027] Reverse transcription uses the RNA PCR Kit purchased from Takara, and the steps ar...

Embodiment 2

[0036] Example 2 Analysis of transglycosylation activity of β-glucosidase Cel1b

[0037] Add 100μg of β-glucosidase Cel1b prepared in Example 1 to 300μl reaction system, 20%-40%(w / v) glucose or 10%-20%(w / v) cellobiose, the reaction buffer is 50 mM sodium phosphate buffer (pH 7.0), reaction product without enzyme as a control, react at 45°C for 24 hours. After the product was boiled for 10 min, centrifuged to collect the supernatant, mixed with bed resinTMD-8 (Sigma, USA) by vortexing for 1 min, centrifuged to collect the supernatant, and the supernatant was obtained with the RID-10A refractive index detector and Bio- The product was analyzed by LC-10AD HPLC (Shimadzu, Japan) of Rad Aminex HPX-42A carbohydrate column. Sugar column temperature is 75℃, use ddH 2 O is eluted at a flow rate of 0.4 ml / min. The product is analyzed by HPLC, such as figure 2 with image 3 , Cellobiose is produced when glucose is used as the substrate) Cellotriose and cellotetraose are produced when cel...

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Abstract

The invention relates to beta-glucosidase Cel1b, and expression gene and application thereof, and belongs to the technical field of bioengineering. The invention provides a cel1b gene expressing beta-glucosidase Cel1b, and the cel1b gene has a nucleotide sequence shown a sequence SEQ ID NO.1. The invention also provides a beta-glucosidase Cel1b with an amino acid sequence shown as a sequence SEQ ID NO.2. The beta-glucosidase Cel1b of the invention can be used in industrial synthesis of expensive cello-oligosaccharide and be further applied to research on cellulase hydrolysis mechanism and utilization of cellulose by microbes. Besides, the cello-oligosaccharide synthesized by an in vitro biological method can be used as a functional food or food additive as well as sweetener for diabetes patients; and the cello-oligosaccharide can also be applied to cosmetic industry and pharmaceutical industry and has a wide application prospect.

Description

Technical field [0001] The invention relates to β-glucosidase Cel1b and its expressed genes and applications, and belongs to the technical field of bioengineering. Background technique [0002] There are abundant lignocellulose resources on the earth, which have huge application potential in the production of renewable alternative energy and basic compounds. There are many microorganisms that can effectively degrade and transform lignocellulose in nature, and their degradation process of lignocellulose constitutes an important aspect of the earth's carbon cycle. For example, Trichoderma reesei is the most important representative strain. It mainly degrades cellulose by secreting cellulase, including exoglucosidase, endoglucosidase, β-glucosidase, etc. Most of the cellulase produced by fungi are induced enzymes. Adding inducers and regulating cellulase synthesis pathways are effective methods to increase cellulase activity. It is now generally accepted that certain soluble oligo...

Claims

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Application Information

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IPC IPC(8): C12N15/56C12N9/42C12N15/63C12N1/15C12N1/19C12N1/21C12N5/10C12P19/14
Inventor 周庆新刘巍峰陈冠军徐金涛吕新星
Owner SHANDONG UNIV
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