Mutant of pectin lyase from Aspergillus niger and viscosity reducer of corn syrup in alcohol fermentation production

A technology of pectin lyase and mutants, which is applied in the field of pectin lyase mutants of Aspergillus niger and syrup viscosity reducers in corn alcohol fermentation production, which can solve the problem of raising drying temperature, prolonging high temperature residence time, and reducing corn Fuel ethanol enterprise profit and other issues, to achieve the effect of reducing steam consumption, excellent enzyme stability, excellent catalytic activity

Active Publication Date: 2019-02-22
NANJING NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the low concentration of syrup, a large amount of water is brought in, which greatly increases the energy consumption of feed drying steam, raises the drying temperature, prolongs the high temperature residence time, destroys a large amount of nutrients, and produces a large amount of Maillard reaction, which makes DDGS darker. , it is easy to have a burnt smell, which affects the palatability of animals, and finally affects the grade and price of DDGS feed, reducing the profits of corn fuel ethanol enterprises

Method used

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  • Mutant of pectin lyase from Aspergillus niger and viscosity reducer of corn syrup in alcohol fermentation production
  • Mutant of pectin lyase from Aspergillus niger and viscosity reducer of corn syrup in alcohol fermentation production
  • Mutant of pectin lyase from Aspergillus niger and viscosity reducer of corn syrup in alcohol fermentation production

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Experimental program
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Effect test

Embodiment 1

[0028] Preparation and properties of embodiment 1 Aspergillus niger pectin lyase mutant

[0029] 1. Sequence of the mutant

[0030] The amino acid sequence of Aspergillus niger (Aspergillus niger) pectin lyase is shown in SEQ ID NO: 1 (this sequence consists of 137 and 204 in the original sequence of Aspergillus niger pectin lyase (GenBank: AIE38009.1) of NCBI X is taken as K and D respectively), and the cDNA sequence of the gene is shown in SEQ ID NO:2.

[0031] After analysis by the molecular simulation software PROPKA software, the lysine (K) at position 241 of the amino acid sequence of Aspergillus niger pectin lyase was replaced with methionine (M) to obtain mutant K241M, and the codons were replaced accordingly. For Escherichia coli, using http: / / www.jcat.de Codon optimization was performed on the website, and finally the coding gene sequence of the mutant K241M was obtained, as shown in SEQ ID NO:3.

[0032] 2. Preparation of pectin lyase mutants from Aspergillus ni...

Embodiment 2

[0041] The preparation of embodiment 2 viscosity reducer

[0042] Viscosity reducers of the following compositions were prepared respectively. The parts in this embodiment are parts by mass. In this example, the original Aspergillus niger pectin lyase and the mutant K241M obtained the fermentation liquid according to the method in Example 1 respectively, and suspended the thallus in phosphate buffer to make OD 600Both were suspensions of 4.5, and the crude enzyme solution was obtained by ultrasonic crushing. The enzyme activities of the crude enzyme solution were 6.47IU / ml and 9.8IU / ml respectively. Purify according to the method in the literature (Cheng Lifeng et al., Construction of a pectin lyase gene (pel) expression system and the enzymatic properties of its expression product, Journal of Agricultural Biotechnology, 2013, 21 (5): 546-553) The pectin lyase was freeze-dried, and the purity of the purified original Aspergillus niger pectin lyase and the mutant K241M were f...

Embodiment 3

[0050] Application of embodiment 3 viscosity reducer

[0051] The technological process of corn alcohol fermentation is: corn flour—pulp mixing—adding enzyme preparation and yeast—raw meal fermentation—distillation—dehydration—fuel ethanol. The addition amount of each substance in the fermentation is 2kg of corn flour, 0.1-0.5% of yeast, 20-80U / g corn flour of alpha-amylase, and 100-200U / g corn flour of glucoamylase. Fermentation conditions: material-to-water ratio 1:2-1:5, constant temperature 28-35°C, stirring speed 100-200r / min, fermentation without cooking for 3-6 days. After the fermentation is finished, the fuel ethanol is distilled out, and the distillation waste mash is filtered to obtain a clear liquid. Concentrate the supernatant to a syrup with a concentration (mass percentage of dry matter, the same below) of 29%. Viscosity reducing agent 1 and comparison viscosity reducing agent 1, viscosity reducing agent 2 and comparison viscosity reducing agent 2 in Example 2...

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Abstract

The invention provides an aspergillus niger pectin lyase mutant and a viscosity reducer for syrup during corn ethanol fermentation production, and relates to the field of biotechnologies. Lysine at the 241st positions of amino acid sequences of aspergillus niger pectin lyase is replaced by methionine to obtain the mutant. The invention further provides an encoding gene of the mutant. A method for preparing the mutant includes fermenting Escherichia coli with the encoding gene of the mutant to obtain the mutant. The viscosity reducer for the syrup during corn ethanol fermentation production is a mixture of the mutant and one or two types of cellulase, xylanase, beta-glucanase, mannanase, glutamine transaminase and lipase. The aspergillus niger pectin lyase mutant, the viscosity reducer and the encoding gene have the advantages that the aspergillus niger pectin lyase mutant is excellent in enzyme stability and catalytic activity, and the method for preparing the aspergillus niger pectin lyase mutant is simple; the viscosity of the syrup can be quickly and efficiently reduced by the viscosity reducer during corn ethanol fermentation production under the condition of low energy consumption.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a mutant of Aspergillus niger pectin lyase and a viscosity reducer for syrup in corn alcohol fermentation production. Background technique [0002] There are two types of corn distillers grain protein feed products on the market: one is DDG (Distillers Dried Grains), which is a feed obtained by simply filtering corn distillers grains, drying the filter residue, draining the filtrate, and drying the filter residue alone; One is DDGS (Distillers Dried Grains with Solubles) feed, that is, dried distiller's grains containing soluble solids, which is obtained by drying and concentrating the filtrate and then mixing and drying the filter residue. The energy and total nutrients of the latter are significantly higher than the former. [0003] The distiller's waste mash after corn alcohol fermentation is centrifuged or plate-and-frame filtered to obtain wet distiller's grains (WDG) and super...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/88C12N15/60C12N15/70C12N9/42C12N9/10C12N9/20C12N9/96A23K10/38
CPCC12N9/1044C12N9/20C12N9/2437C12N9/2448C12N9/248C12N9/2488C12N9/88C12Y203/02013C12Y301/01003C12Y302/01073C12Y402/02002Y02P60/87
Inventor 黄和俞亚东李秀娟
Owner NANJING NORMAL UNIVERSITY
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