Design and preparation for mannose with high substrate affinity

A technology of mannanase and mannobiose, which is applied in the field of genetic engineering and protein expression, can solve the problems of molecular transformation research that has not been reported, and achieve the effects of large-scale industrial production, high catalytic efficiency and strong substrate affinity

Inactive Publication Date: 2013-03-27
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, although there are some literatures and patent reports on the cloning and expression of β-mannanase gene, the research on the molecular modification of mannanase based on rational design, especially the molecular modification of mannanase affinity no reports

Method used

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  • Design and preparation for mannose with high substrate affinity
  • Design and preparation for mannose with high substrate affinity
  • Design and preparation for mannose with high substrate affinity

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] The construction of embodiment 1 mutant enzyme gene and its expression plasmid

[0027] Construction of Mutant Enzyme Gene man5A Using Large Primer PCR Technology Y111F 、man5A Y111F: AMan5A-F and Y111FA, Man5A-F and Y115F were used as primers and pUCm-T-man5A as a template for PCR (94°C 4min; 94°C 30s, 55°C 30s, 72°C 30s, 30 cycles; 72°C 10min), the PCR product was analyzed by 1% agarose gel electrophoresis, and the target band was recovered by tapping the rubber to obtain a large primer; the product recovered from the first round of PCR tapping and Tman5A-R were used as primers for large primer PCR (94°C 4min; 94°C 30s , 55°C for 30s, 72°C for 70s, 30 cycles; 72°C for 10min), the PCR product was analyzed by 1% agarose gel electrophoresis, the target band was recovered by tapping the gel and ligated with pUCm-T (pUCm-T-man5A Y111F , pUCm-T-man5A Y115F ), transformed into JM109 competent cells, and sent to Shanghai Sangon for sequencing. Will sequence the correct pUC...

Embodiment 2

[0028] Embodiment 2GS115 / man5A Y111F 、GS115 / man5A Y115F Construction, expression, product purification and activity determination of

[0029] pPIC9K-man5A with Sal I Y111F , pPIC9K-man5A Y115F Perform linearization, perform electrotransformation and screening according to the Pichia expression manual, and obtain high-copy Pichia recombinant GS115 / man5A Y111F 、GS115 / man5A Y115F . The genetically engineered bacterium was induced to express with 1.0% methanol for 96 hours, and the activity of mannanase in the fermented liquid was measured by DNS method to be 80IU / mL and 60IU / mL respectively. The supernatant after centrifugation of the fermentation broth is the crude enzyme solution of the mutant enzyme, which is salted out with ammonium sulfate (adding solid ammonium sulfate to a saturation of 75%), and then subjected to ion exchange chromatography with DEAE-Sepharose Fast Flow and SephadexG-75 gel filtration chromatography, after purification, it was detected as a single b...

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Abstract

The invention discloses a design and preparation for mannose with high substrate affinity. The invention provides a method for designing and constructing AusMan5AY111F and AusMan5AY115F mutant enzymes with high substrate affinity. Mutation sites are finally determined by sequence homology analysis, homologous modeling, and calculating of binding energy; and the corresponding genes obtained by megaprimer PCR (Polymerase Chain Reaction) are named as Ausman5AY111F and man5AY115F. The invention further discloses a method for constructing, efficiently expressing and purifying mutant enzyme engineering bacteria. The prepared mutant enzymes have the characteristics of high substrate affinity and high catalytic efficiency, large industrial potential productivity and a high economic value.

Description

technical field [0001] The present invention relates to the field of genetic engineering and protein expression, especially AusMan5A Y111F and AusMan5A Y115F The construction and expression of the enzyme gene, as well as the research on the properties and preparation of the enzyme. Background technique [0002] β-mannanase (β-1,4-D-mannan mannohydrolase, EC 3.2.1.78) is a kind of enzyme that can degrade β-1,4-mannoside from the interior of the main chain of homomannan and heteromannan Key hydrolases belong to the class of hemicellulase, which widely exists in microorganisms, plants and animals. It can be widely used in the fields of food, medicine, feed, papermaking, printing and dyeing, and petroleum industries. In the field of food and medicine, β-mannanase can produce functional oligosaccharides after acting on mannan, which can effectively reduce human blood sugar and cholesterol levels and benefit the intestinal probiotics of humans and animals Group growth, and the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/56C12N9/42C12N15/10C12R1/66
Inventor 李剑芳胡蝶汪俊卿魏喜换赵梅邬敏辰
Owner JIANGNAN UNIV
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