Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

High-efficiency preparation method of beta-amylase

An amylase and seed technology, applied in the field of enzyme engineering and bioengineering, can solve the problems of low beta-amylase activity, poor heat resistance and high cost, and achieve the effects of high enzyme activity level, good thermal stability and low cost

Active Publication Date: 2012-11-07
江苏锐阳生物科技有限公司
View PDF2 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, the β-amylase produced by microbial fermentation in my country has low activity, poor heat resistance, high cost, and relatively little research

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • High-efficiency preparation method of beta-amylase
  • High-efficiency preparation method of beta-amylase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] The sweet potato RNA that embodiment 1 Trizol method extracts

[0059] (1) Clean the sweet potato, cut it into small pieces, grind it into powder in liquid nitrogen, transfer about 50~100 mg sample into a 1.5 mL centrifuge tube, add 1 mL Trizol reagent, invert and mix well to suspend the sample in the reagent middle.

[0060] (2) After the suspension was left at room temperature for 5 minutes, 0.2 mL of chloroform was added, the centrifuge tube was shaken vigorously for 15 seconds, left at room temperature for 5 minutes, and then centrifuged at 12,000 rpm for 10 minutes at 4°C.

[0061] (3) Carefully pipette the upper aqueous phase into a new centrifuge tube, add 0.5 mL of isopropanol, mix well, place at room temperature for 10 min, and then centrifuge at 12,000 rpm for 10 min at 4°C.

[0062] (4) Discard the supernatant, add 1 mL of 75% ethanol to wash the precipitate, and centrifuge at 12,000 rpm for 5 min at 4°C.

[0063] (5) Discard the supernatant, and dry the pe...

Embodiment 2

[0064] Example 2 Synthesis of cDNA and amplification of β-amylase gene

[0065] (1) Synthesis of cDNA by reverse transcription

[0066] Reaction system (20 μL): 1 μg RNA, reverse transcription primer 10 mM oligdT-18 1 μL, 5×Buffer 4 μL, 10M dNTPs 1 μL, M-MLV reverse transcriptase 1 μL, remaining volume with RNase-free water make up.

[0067] Reaction conditions: first add RNA and reverse transcription primers to the reaction tube, make up the volume to 10 μL with RNase-free water, 70°C, 5 min, and place on ice for 5 min; then add the remaining reagents, make up the volume to 20 μL with RNase-free water, 42°C, 60 min.

[0068] (2) PCR amplification containing the complete β-amylase gene BBA, the primers used are:

[0069] BBA-F: 5′-a agatct ccatggctccaatccccggt-3′

[0070] BBA-R: 5′-a agatct gctcctccttcaccttcag-3′

[0071] Amplified using Takara's ExTaq DNA polymerase

[0072] reaction system:

[0073] Volume (μL) Sterilized double distilled water 33...

Embodiment 3

[0076] Example 3 Construction of Secreted Expression Vector pPIC9K-(A / B / C / D)-OBBA

[0077] Using 10 pg of the Teasy-BBA plasmid as a template, PCR amplifies the target fragment 0BBA, and the primers used are:

[0078] 0BBA-F: 5′-atggctccaatccccggt-3′,

[0079] 0BBA-R: 5'-tcaatcaaacgggtttgagccatc-3';

[0080] Reaction system and reaction condition are the same as embodiment 2.

[0081] The amplified product was the 1500 bp open reading frame sequence of the β-amylase gene except the signal peptide coding region.

[0082] The target gene OBBA and Pichia pastoris secreted expression vector pPIC9K-A were respectively used Bgl II and Bam HI was subjected to double enzyme digestion, and reacted overnight at 37°C; after the plasmid digestion product and PCR product were purified with a purification kit, T 4 DNA ligase was ligated at 4°C for 14 h. Transform the ligation product into Escherichia coli JM109, smear the LB plate containing 50 μg / mL kanamycin, select positive clon...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

PropertyMeasurementUnit
pore sizeaaaaaaaaaa
Login to View More

Abstract

The invention relates to a high-efficiency preparation method of beta-amylase, belonging to the technical fields of enzyme engineering and biological engineering. The preparation method comprises the following steps: respectively positioning a sweet potato-derived beta-amylase gene 0BBA or a signal peptide-deleted beta-amylase gene 28BBA in four different secretion signal peptides (pPIC9K-A, pPIC9K-B, pPIC9K-C or pPIC9K-D) to construct 8 different recombinant plasmids, converting into two Pichia pastoris (GS115 or KM71) incapable of producing beta-amylase through an electroporation method, and screening to obtain the Pichia pastoris CCTCC NO:M2011014 which is converted from KM71 and contains pPIC9K-A-0BBA; carrying out high-density fermentation on the Pichia pastoris CCTCC NO:M2011014, wherein the enzyme activity of the fermentation liquid can be 500U / mL; and carrying out centrifugation, ultrafiltration, microfiltration and freeze-drying to obtain the beta-amylase, wherein the protein concentration of the finished product is up to 3-6g / L, and the enzyme activity is up to 2000U / g. The invention can be used for the high-efficiency production of beta-amylase for starch processing, sugar making and food processing industries.

Description

technical field [0001] The invention relates to a method for producing beta-amylase by Pichia pastoris genetically engineered bacteria, and belongs to the technical fields of enzyme engineering and bioengineering. Background technique [0002] β-amylase (EC 3.2.1.2), that is, (1→4)-α-glucan maltohydrolase, is an exonuclease, which starts from the non-reducing end of starch and hydrolyzes sequentially by maltose unit, simultaneously The Walden transposition reaction changes the product from α-type to β-type maltose. This enzyme cannot hydrolyze the α-1,6 glucosidic bond at the branch of starch, and the decomposition of starch will stop at 2~3 glucose residues before the 1,6 bond, so the product of amylose decomposition is mainly maltose, and the decomposition of branch The products of amylopectin are mainly maltose and macromolecular β-limit dextrin. β-amylase is widely found in higher plants such as barley, wheat, corn, soybean, and sweet potato, and some microorganisms ca...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/19C12N15/56C12N15/81C12N9/26C12R1/84
Inventor 王正祥
Owner 江苏锐阳生物科技有限公司
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com