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Method for producing ethanol by synchronously performing steam-exploded pennisetum alopecuroides batch feeding and saccharification fermentation

A technology of simultaneous saccharification and fermentation and pennisetum, applied in the field of fermentation, can solve the problems of unfavorable industrial production, lack of direct degradation of lignocellulose substrate performance, and few research reports

Inactive Publication Date: 2020-11-20
CAPITAL NORMAL UNIVERSITY
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AI Technical Summary

Problems solved by technology

[0009] The above-mentioned studies on the hydrolysis and fermentation of chimeric cellosomes to produce ethanol from cellulose have the following problems: 1. The substrate of cellosome hydrolysis and fermentation is mainly cellulose or hemicellulose, and the substrate is hydrolyzed into glucose or xylose , and then fermented by Saccharomyces cerevisiae to produce ethanol, there is no research on lignocellulose as substrate
2. The types of enzymes assembled in the cellulosome structure are relatively single, and the common enzymes are cellulase or hemicellulase, which do not have the ability to directly degrade lignocellulosic substrates
3. The structure of cellulosomes is relatively simple, which can neither be used to increase the loading capacity of enzymes nor enhance the synergistic effect between enzyme molecules
4. The cellosome assembly method is mainly to incubate and assemble the cellosome protein expressed by Escherichia coli and the cell surface display strain of Saccharomyces cerevisiae. This process is complicated to operate and the assembly efficiency is low, which is not conducive to industrial production
[0011] At present, there are few research reports on the use of cellulosomes in degrading lignocellulose to ferment ethanol

Method used

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  • Method for producing ethanol by synchronously performing steam-exploded pennisetum alopecuroides batch feeding and saccharification fermentation
  • Method for producing ethanol by synchronously performing steam-exploded pennisetum alopecuroides batch feeding and saccharification fermentation
  • Method for producing ethanol by synchronously performing steam-exploded pennisetum alopecuroides batch feeding and saccharification fermentation

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Embodiment 1: Construction of recombinant Saccharomyces cerevisiae strain

[0053] 1.1 According to the cohesion module gene sequence published by NCBI (the cohesion module gene and its gene sequence numbers are: CipA (GI: 125972525), CipC (GI: 11056042) and ScaB (GI: 13277318)). The PCR reaction primers were designed, and the primer sequences are shown in Table 1.

[0054] Table 1: Primer sequences for constructing the recombinant vector pYD1-PGK-Aga2-ScafI

[0055]

[0056]The genomes of Clostridium cellulolyticum, Clostridium thermocellum and Ruminococcus flavefaciens were respectively used as templates for gene amplification to amplify the cohesion module gene of the scaffold protein ScafI, and passed the Over- Amplified by lap PCR method. The Over-lap PCR reaction system is: 4.0 μL dNTPs, 2.0 μl Primer CipA-F, 2.0 μL Primer CipC-R, 10.0 μL 5×Q5 Reaction Buffer, 0.5 μL Q5 DNA Polymerase (NEB Co., Ltd.), CipA, CipC and Add 1.0 μL of each ScaB gene to 50.0 μL wi...

Embodiment 2

[0079] Example 2: Construction of self-assembled cellulosomes in Saccharomyces cerevisiae

[0080] In YPD (1% yeast extract, 2% peptone, 2% glucose, 10mmoL CaCl 2 , pH is 4.5~6.5) culture medium respectively inserts 1% (v / v) Saccharomyces cerevisiae Y5-ScafI, Y6-1 and Y6-2 cells constructed in embodiment 1, and expresses in embodiment 1 respectively Recombinant strains Y6-EGII, Y6-CBHII, Y6-BGLI, Y6 constructed by transforming Saccharomyces cerevisiae Y6 cells with cellulase (EGII, CBHII and BGLI) and secreted expression plasmids expressing hemicellulase (XylA and XynII) respectively -XylA and Y6-XynII were cultured at 30° C., 150 rpm for 24 hours. Assembly verification was performed using flow cytometry and confocal immunofluorescence microscopy imaging.

[0081] Such as figure 1 As shown, Saccharomyces cerevisiae Y5 cells transformed with pYD1 empty vector were used as a negative control (NegativeControl). The C-terminus of ScafI, the primary scaffold protein of cellulos...

Embodiment 3

[0082] Example 3: Determination of Enzyme Activity of Cellulases and Hemicellulases

[0083] (1) Enzyme activity assay of EG II and CBH II: After culturing recombinant yeast cell Y6-EGII or Y6-CBHII in SD liquid medium (Meitan, M300-03) for 48 hours, take 500 μl of crude enzyme solution in a test tube, add 1 mL of substrate (EGII substrate is 1 % carboxymethyl cellulose (Carboxymethyl Cellulose, CMC), prepared with 0.2mM citric acid buffer, pH 5.0; the substrate of CBHII is 1% PASC, prepared with 0.2mM sodium acetate buffer, pH 5.0), vortex After shaking and mixing, react in a water bath at 50°C for 30 minutes, then add 2.5 mL of DNS chromogenic solution, boil for 5 minutes, cool it rapidly in ice water, and add ultrapure water to make up to 25 mL. Absorbance was measured at 540 nm by a UV-Vis spectrophotometer. Three samples were tested respectively, and the average value of the absorbance value was taken. In the control group, first add DNS chromogenic solution to warm b...

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Abstract

The invention discloses a method for producing ethanol by synchronously performing steam-exploded pennisetum alopecuroides batch feeding and saccharification fermentation. The method for producing theethanol by synchronously performing steam-exploded pennisetum alopecuroides batch feeding and saccharification fermentation comprises the following steps: (1) constructing recombinant yeast cells which can co-metabolize xylose and glucose and are anchored with primary scaffold protein Scaf I; (2) assembling cellulosomes derived from cellulase and hemicellulase on the surfaces of the yeast cells so as to obtain corresponding recombinant yeast cells; (3) carrying out co-culture on the recombinant yeast cells in the step (1) and the step (2), and forming recombinant yeast cells containing composite functional cellulosomes through self-assembly of the recombinant yeast cells; and (4) inoculating the recombinant yeast cells obtained in the step (3) into a fermentation culture medium, and carrying out fermenting by using steam exploded pennisetum alopecuroides as a unique carbon source according to a batch feeding method to produce the ethanol. By utilization of the method disclosed by theinvention, the concentration of the ethanol obtained by fermentation for 96 hours can reach 2.65 g / L.

Description

technical field [0001] The invention relates to the field of fermentation, in particular to a method for producing ethanol by steam-exploding Pennisetum batch-type fed-batch synchronous saccharification and fermentation. Background technique [0002] Biofuel ethanol mainly refers to a green fuel with a volume concentration of more than 99% and an octane number of up to 115 produced from biomass. It is usually mixed with gasoline in a certain proportion and can be used as a good oxygenate and blending agent for gasoline. agent, thereby improving the combustion performance of gasoline, while reducing the emission of particulate deposits and carbon dioxide in automobile exhaust [1] . [0003] The current production of biofuel ethanol in the world mainly uses sugar and starch as raw materials, and with the increase in demand for clean energy, the second-generation biofuel ethanol using lignocellulose as raw material can solve the problem of biofuel ethanol using grain as raw ma...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P7/10C12N15/81C12N15/31C12N15/56C12N1/19C12R1/865
CPCC07K14/195C07K14/33C12N9/2437C12N9/2477C12N15/81C12P7/10Y02E50/10
Inventor 田沈杨秀山杜济良
Owner CAPITAL NORMAL UNIVERSITY
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