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

Pullulanase mutant, engineering bacterium and application of pullulanase mutant

A technology of pullulanase and pullulanase protein is applied in the field of preparing pullulanase with improved thermal stability and acid resistance, which can solve the problems of poor stability and low catalytic activity, and achieve good catalytic performance and industrial properties. Effect

Pending Publication Date: 2022-07-29
ZHEJIANG UNIV OF TECH
View PDF0 Cites 1 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to provide a pullulanase PulAR mutant, engineering bacteria and application thereof, improve the catalytic activity, thermal stability and acid resistance stability of pullulanase PulAR, and solve the problem of wild-type pullulanase PulAR in high temperature, Low catalytic activity and poor stability under acidic conditions

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
  • Pullulanase mutant, engineering bacterium and application of pullulanase mutant
  • Pullulanase mutant, engineering bacterium and application of pullulanase mutant
  • Pullulanase mutant, engineering bacterium and application of pullulanase mutant

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1: Construction of wild-type pullulanase recombinant bacteria

[0024] According to the gene (KY273924) of type I pullulanase derived from thermoanaerobic bacillus (Anoxybacillus sp.) AR-29 in Genbank, the cloning primers of pullulanase were designed as shown in Table 1, and the Fragment and inserted into the vector pET-32a(+) between EcoR V and Xho I restriction site to obtain recombinant plasmid pET-32a(+)-PulAR (nucleotide sequence shown in SEQ ID NO.2, encoding The amino acid sequence of the protein is shown in SEQ ID NO.1), the recombinant plasmid pET-32a(+)-PulAR with correct sequencing was transformed into E.coli BL21(DE3), and a single colony was picked for sequencing to obtain recombinant bacteria E. coli BL21(DE3) / pET-32a(+)-PulAR, namely wild-type E. coli BL21(DE3) / pET-32a(+)-PulAR.

[0025] SEQ ID NO.1

[0026] MYEVFSSLILKTNEKMGLFILGGANLLTVHRTFEAYLDTMTVITILIPKSYHSGMVGNFIIEKPNGERCQLQVAKREDLWTSIKYECVIDFAVEIGRRYLIYDDHGAFTDLQIGAVIRTAEFDEQFYYEGNDLGITYTP...

Embodiment 2

[0029] Example 2: Construction and screening of pullulanase mutants

[0030] 1. Construction of pullulanase mutants

[0031] According to the gene sequence of pullulanase PulAR shown in SEQ ID NO.2, design and synthesize mutant primers (Table 1) introducing mutations Q355, A365, T399, V401, Y491, H499 and T504, using the obtained in Example 1 The recombinant plasmid pET-32a(+)-pulAR was used as the template, the whole plasmid was amplified by PCR, transformed into E. coli E.coli BL21(DE3), sequenced and identified, and the single mutant E.coli BL21(DE3)-PulAR was obtained -Q355H, E.coli BL21(DE3)-PulAR-A365V, E.coli BL21(DE3)-PulAR-T399S, E.coli BL21(DE3)-PulAR-V401T, E.coli BL21(DE3)-PulAR-V401C , E.coli BL21(DE3)-PulAR-Y491V, E.coli BL21(DE3)-PulAR-H499A and E.coliBL21(DE3)-PulAR-T504V.

[0032] PCR amplification system: 1 μL forward primer (100 μM), 1 μL reverse primer (100 μM), 12.5 μL 2× Phanta buffer, 0.5 μL dNTP mix (10 mM each), 1 μL plasmid template, 0.5 μL DNA poly...

Embodiment 3

[0046] Example 3: Combinatorial Mutation and Purification of Pullulanase Protein

[0047] 1. Combination mutation

[0048] The beneficial mutations A365V, V401C, T504V and H499A of the pullulanase obtained in Example 2 were subjected to combined mutation research, and combined mutants PulAR-A365V / V401C, PulAR-A365V / V401C / T504V, PulAR-A365V / V401C / T504V / H499A, as follows:

[0049] (1) Construction of combined mutant PulAR-A365V / V401C

[0050] Using the plasmid pET-32a-PulAR-A365V of the single mutant E.coli BL21(DE3)-PulAR-A365V of Example 2 as a template, and using Q355H(F) and Q355H(R) in Table 2 as primers, Example 2 was used Methods Whole plasmid PCR was performed, and the mutant PulAR-A365V / V401C was obtained by screening.

[0051] (2) Construction of combined mutant PulAR-A365V / V401C / T504V

[0052] Taking the above-mentioned plasmid pET-32a-PulAR-A365V / V401C of the constructed combined mutant E.coli BL21(DE3)-PulAR-A365V / V401C as a template, and taking V401C(F) and V4...

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

No PUM Login to View More

Abstract

The invention discloses a pullulanase mutant, an engineering bacterium and application of the pullulanase mutant and the engineering bacterium. The pullulanase mutant is obtained by performing single mutation or multiple mutations on 365th, 401th, 504th or 499th amino acids of an amino acid sequence shown as SEQ ID NO.1. The invention further discloses a preparation method of the pullulanase mutant. The catalytic activity, the thermal stability and the acid-resistant stability of the pullulanase mutant constructed by the invention are all improved, wherein the catalytic efficiency of the mutant PulAR-A365V-401C-H499A-T504V is respectively improved by 6.6 times and 9.6 times compared with that of wild pullulanase PulAR under the conditions that the pH is 5.0, the pH is 6.0 and the temperature is 60 DEG C; the half-life periods at 60 DEG C and 65 DEG C are respectively increased by 2.6 and 3.1 times compared with those of the wild pullulanase, and the half-life periods at pH 4.5 and pH 5.0 are respectively increased by 1.6 and 1.8 times compared with those of the wild pullulanase.

Description

(1) Technical field [0001] The present invention relates to a pullulanase, in particular to a pullulanase PulAR mutant constructed by site-directed mutagenesis and combined mutagenesis, and used to prepare a pullulanase with improved thermostability and acid resistance. (2) Background technology [0002] Pullulanase (EC 3.2.1.41) is a starch debranching enzyme that can specifically cleave α-1,6 in pullulan, soluble starch, amylopectin and some oligosaccharides Glycosidic bond. Pullulanase is widely used in the saccharification step, and works synergistically with saccharification enzyme or β-amylase to improve saccharification efficiency and reduce the usage of saccharification enzyme or β-amylase, thereby increasing production and reducing production costs. [0003] In order to maximize the synergistic effect between pullulanase and saccharification enzyme or β-amylase, pullulanase, saccharification enzyme and β-amylase should have the same working temperature. In industr...

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
IPC IPC(8): C12N9/44C12N15/70C12N1/21C07K1/14C07K1/22C07K1/34C07K1/36C12P19/14C12P19/00C12R1/19
CPCC12N9/2457C12N15/70C12N1/20C12P19/14C12P19/00
Inventor 王亚军李树芳徐沈远张伟郑裕国
Owner ZHEJIANG UNIV OF TECH
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