Mutant of cellulose exocellobiohydrolase CBH I and viscosity reducer of syrup in corn fuel ethanol fermentation production

An exoglucanase and cellulose technology, applied in fermentation, biofuel, enzymes, etc., can solve the problems of increasing energy consumption of feed drying steam, dark color of DDGS, destruction of nutrients, etc., and achieve excellent enzyme stability and catalytic activity, reducing steam consumption, and improving the first-grade product rate

Inactive Publication Date: 2015-11-04
NANJING TIANKAI BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because the concentration of syrup is not high, a large amount of water will be brought in, which will greatly increase the energy consumption of feed drying steam, increase the drying temperature, prolong the high temperature residence time, destroy a large amount of nutrients, produce a large amount of Maillard reaction, and make DDGS color It is darker and easily has a burnt smell, which affects the palatability of raised animals, affects the grade and price of DDGS feed, and thus affects the profits of corn fuel ethanol enterprises
[0005] In the prior art, there is no method for low energy consumption, rapid and efficient viscosity reduction of syrup in corn fuel ethanol fermentation production

Method used

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  • Mutant of cellulose exocellobiohydrolase CBH I and viscosity reducer of syrup in corn fuel ethanol fermentation production
  • Mutant of cellulose exocellobiohydrolase CBH I and viscosity reducer of syrup in corn fuel ethanol fermentation production

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Effect test

Embodiment 1

[0028] Example 1 Preparation and Properties of Cellulose Exoglucanase CBH I Mutant

[0029] 1. Sequence of the mutant

[0030] The amino acid sequence of Trichoderma reesei cellulose exoglucanase CBH I is shown in SEQ ID NO:1, and the gene cDNA sequence is shown in SEQ ID NO:2.

[0031] After analysis by the molecular simulation software PROPKA software, the tyrosine (Y) at position 68 of the amino acid sequence of Trichoderma reesei cellulosic exoglucanase CBH I was replaced with glycine (G) to obtain mutant Y68G, and the corresponding replacement Codon (substitute GGT for TAC at positions 202-204 in SEQ ID NO:2). For Escherichia coli, the website http: / / www.jcat.de was used for codon optimization, and finally the coding gene of mutant Y68G (SEQ ID NO: 3) was obtained. In addition, the tyrosine (Y) at position 68 of the amino acid sequence of Trichoderma reesei cellulose exoglucanase CBH I was replaced with alanine (A) to obtain mutant Y68A, and the mutation was obtained by...

Embodiment 2

[0044] The preparation of embodiment 2 viscosity reducer

[0045] Viscosity reducers of the following compositions were prepared respectively. The parts in this embodiment are parts by mass. The original cellulosic exoglucanase CBH I in this example, two kinds of mutants Y68G and Y68A, respectively obtain the crude enzyme liquid according to the method in Example 1, then according to Document 1 (Pan Feng et al., Usami Aspergillus Y- 26 The purification and enzymatic properties of cellulase, the method in Nanjing University of Science and Technology, 2001) method to purify each cellulosic exoglucanase and freeze-drying, the specific activity of cellulosic exoglucanase CBH I is 21500U / g, the specific activity of mutant Y68G is 39600U / g, and the specific activity of Y68A is 38400U / g. Information on the rest of the enzymes used as table 2 shown.

[0046] Table 2 Information on enzymes used in complex viscosity reducers

[0047] Enzyme name factory product nu...

Embodiment 3

[0054] Application of embodiment 3 viscosity reducer

[0055] The technological process of corn fuel ethanol 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 reduction agent 1 and comparison viscosity reduction agent 1, viscosity reduction agent 4 and comparison viscosity reduction agent 2 in ...

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Abstract

The invention provides a mutant of cellulose exocellobiohydrolase CBH I and viscosity reducer of syrup in corn fuel ethanol fermentation production and relates to the field of biotechnology. The mutant of the cellulose exocellobiohydrolase CBH I is obtained by replacing the 68th tyrosine of the amino acid sequence of the cellulose exocellobiohydrolase CBH I with glycine or alanine. The viscosity reducer is a mixture of the mutant of the cellulose exocellobiohydrolase CBH I and one or more than two of pectinase, xylanase, beta-glucanase, mannase, glutamine transaminase and lipase. The mutant of the cellulose exocellobiohydrolase CBH I is great in enzyme stability and catalytic activity, and the preparation method is simple and efficient. By adopting the viscosity reducer, viscosity reduction can be conducted on the syrup in the corn fuel ethanol fermentation production rapidly and efficiently 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 cellulose exoglucanase CBH I and its application in reducing the viscosity of the clear liquid concentrate in starch fermentation ethanol. Background technique [0002] For a long time, fossil energy has been one of the main energy sources that human beings rely on. However, fossil energy is a non-renewable resource, which is currently being exhausted after being exploited and used in large quantities for a long time; in addition, the long-term use of fossil resources has also brought about increasingly serious environmental pollution problems. In view of the above reasons, countries around the world have been actively looking for various renewable and clean energy sources that can replace fossil resources, among which fuel ethanol is one of the most popular renewable clean energy sources. [0003] However, since 2014, due to the 60% drop in international crude oil price...

Claims

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Application Information

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IPC IPC(8): C12N9/42C12N9/46C12N9/10C12N9/20C12N9/88C12N15/56C12N15/70C12P7/10
CPCC12N9/1096C12N9/20C12N9/2437C12N9/2454C12N9/88C12P7/10Y02E50/10
Inventor 黄和俞亚东李秀娟李干禄
Owner NANJING TIANKAI BIOTECH
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