Method for producing cellooligosaccharides from lignocellulose

Abstract

The invention provides a cellulosome based novel enzyme preparation cello-oligosaccharide preparation method aiming at problems of low efficiency, low yield, high cost and the like in industrial cello-oligosaccharide production in the prior art. The preparation method includes steps of raw material pretreatment, preparation of enzyme preparations, enzymolysis, aftertreatment and the like. Comparedwith an existing technique for producing cello-oligosaccharide from lignocelluloses, the method has advantages that production cost caused by high price of enzymes is reduced; in addition, due to extensive raw material sources and low cost, industrial promotion is technically supported. Further, by adoption of the method, technical problems of (1) serious feedback inhibition caused by cellobioseto cellulosome activity and (2) purification of oligosaccharide subjected to enzymolysis in adoption of cellulosome and production bacterial strains for cello-oligosaccharide preparation are solved. More importantly, compared with the prior art, the method has the advantage of high cello-oligosaccharide yield and conversion rate.

Description

technical field

[0001] The invention relates to the field of microbial fermentation engineering, in particular to a method for preparing cellooligosaccharides from lignocellulosic raw materials. Background technique

[0002] Oligosaccharides are also called oligosaccharides. It is a sugar molecule formed by connecting two or more (generally 2-10) monosaccharide units with glycosidic bonds. Cellulooligosaccharide (cellulooligosaccharide) is a homologous functional oligosaccharide, an oligosaccharide composed of less than 10 glucose molecules connected by β-1,4-glycosidic bonds, and is a product of cellulose degradation. Since there is no digestive enzyme that can degrade β-1,4 glycosidic bonds in the human gastrointestinal tract, the fiber oligosaccharides are not digested and absorbed and directly enter the large intestine for use by bifidobacteria, so the fiber oligosaccharides can be used as functional food or food additives , for intestinal regulation. Cellooligosaccha...

Examples

Embodiment 1

[0028] Example 1: Knockout of exocellulase from Clostridium thermocellum cellulosomes

[0029]Through seamless cloning, the tdk expression cassette (promoter comprising the gapDH gene) and the pyrF expression cassette (comprising the pyrF self-promoter) were cloned into the plasmid pHK (Mohr, G., Hong, W., Zhang, J., Cui, G.-Z., Yang, Y., Cui, Q., et al. (2013) A targetron system for gene targeting in thermophiles and its application in Clostridium thermocellum, PLoS One 8:e69032.) downstream of the antibiotic gene cat, And through the design of primers, NheI and XbaI restriction sites were added between tdk and pyrF expression cassettes, and EagI and MluI restriction sites were added downstream of pyrF for cloning of homology arm fragments, thereby constructing pHK-HR plasmid.

[0030] The cellulosic exonuclease Cel48S (encoded by the nucleic acid sequence 3228088-3230229 in the genome CP002416.1) of the cellulosome of Clostridium thermocellum was selected for targeted knock...

Embodiment 2

[0034] Example 2: Knockout of exocellulase from Clostridium thermocellum cells

[0035] The difference from Example 1 is that the exocellulase Cel9K (encoded by the nucleic acid sequence 2113813-2111293 in the genome CP002416.1) of the cellulosome of Clostridium thermocellum was selected for targeted knockout gene. The upstream homology arm HR-up is the nucleic acid sequence from 2110625 to 2111125 in the Clostridium thermocellum DSM1313 genome (the sequence number in the NCBI database is CP002416.1), the downstream homology arm HR-down is the nucleic acid sequence from 2113814 to 2114314 in the DSM1313 genome, and the middle The homology arm HR-short is the nucleic acid sequence from 2110825 to 2111125 in the DSM1313 genome. Knockout plasmids do not contain the gene of interest. The constructed plasmid was transformed into the DSM1313 mutant strain of pyrF deletion, and the Cel9K knockout strain ΔCel9K was obtained according to the three-step screening method.

Embodiment 3

[0036] Example 3: Modification of exocellulase of Clostridium thermocellum cellulosomes

[0037] The difference from Example 1 is that the knockout plasmid contains the target gene Cel9-48 (encoded by the nucleic acid sequence 1968724 to 1973904 in the genome CP001393.1). The constructed plasmid was transformed into the DSM1313 mutant strain of pyrF deletion, and the Cel9-48 replaced Cel48S strain ΔCel48S::Cel9-48 according to the three-step screening method.

technical field

[0001] The invention relates to the field of microbial fermentation engineering, in particular to a method for preparing cellooligosaccharides from lignocellulosic raw materials. Background technique

[0002] Oligosaccharides are also called oligosaccharides. It is a sugar molecule formed by connecting two or more (generally 2-10) monosaccharide units with glycosidic bonds. Cellulooligosaccharide (cellulooligosaccharide) is a homologous functional oligosaccharide, an oligosaccharide composed of less than 10 glucose molecules connected by β-1,4-glycosidic bonds, and is a product of cellulose degradation. Since there is no digestive enzyme that can degrade β-1,4 glycosidic bonds in the human gastrointestinal tract, the fiber oligosaccharides are not digested and absorbed and directly enter the large intestine for use by bifidobacteria, so the fiber oligosaccharides can be used as functional food or food additives , for intestinal regulation. Cellooligosaccha...

Examples

Embodiment 1

[0028] Example 1: Knockout of exocellulase from Clostridium thermocellum cellulosomes

[0029]Through seamless cloning, the tdk expression cassette (promoter comprising the gapDH gene) and the pyrF expression cassette (comprising the pyrF self-promoter) were cloned into the plasmid pHK (Mohr, G., Hong, W., Zhang, J., Cui, G.-Z., Yang, Y., Cui, Q., et al. (2013) A targetron system for gene targeting in thermophiles and its application in Clostridium thermocellum, PLoS One 8:e69032.) downstream of the antibiotic gene cat, And through the design of primers, NheI and XbaI restriction sites were added between tdk and pyrF expression cassettes, and EagI and MluI restriction sites were added downstream of pyrF for cloning of homology arm fragments, thereby constructing pHK-HR plasmid.

[0030] The cellulosic exonuclease Cel48S (encoded by the nucleic acid sequence 3228088-3230229 in the genome CP002416.1) of the cellulosome of Clostridium thermocellum was selected for targeted knock...

Embodiment 2

[0034] Example 2: Knockout of exocellulase from Clostridium thermocellum cells

[0035] The difference from Example 1 is that the exocellulase Cel9K (encoded by the nucleic acid sequence 2113813-2111293 in the genome CP002416.1) of the cellulosome of Clostridium thermocellum was selected for targeted knockout gene. The upstream homology arm HR-up is the nucleic acid sequence from 2110625 to 2111125 in the Clostridium thermocellum DSM1313 genome (the sequence number in the NCBI database is CP002416.1), the downstream homology arm HR-down is the nucleic acid sequence from 2113814 to 2114314 in the DSM1313 genome, and the middle The homology arm HR-short is the nucleic acid sequence from 2110825 to 2111125 in the DSM1313 genome. Knockout plasmids do not contain the gene of interest. The constructed plasmid was transformed into the DSM1313 mutant strain of pyrF deletion, and the Cel9K knockout strain ΔCel9K was obtained according to the three-step screening method.

Embodiment 3

[0036] Example 3: Modification of exocellulase of Clostridium thermocellum cellulosomes

[0037] The difference from Example 1 is that the knockout plasmid contains the target gene Cel9-48 (encoded by the nucleic acid sequence 1968724 to 1973904 in the genome CP001393.1). The constructed plasmid was transformed into the DSM1313 mutant strain of pyrF deletion, and the Cel9-48 replaced Cel48S strain ΔCel48S::Cel9-48 according to the three-step screening method.