Method for preparing sodium gluconate from lignocellulose

Abstract

The invention provides a method for producing sodium gluconate from lignocellulose agriculture and forestry wastes. The method comprises the following steps: (1) preprocessing: preprocessing a lignocellulose raw material to obtain a lignocellulose substrate; (2) saccharifying: transferring the lignocellulose substrate to a medium in an anaerobic fermentation tank, adding a cellulase preparation, and carrying out a hydrolysis reaction to obtain a glucose-containing sugar solution; (3) roughly preparing: allowing the sugar solution to enter a fixed bed reactor with glucose oxidase and catalase after the concentration of glucose in the sugar solution reaches 10-100 g / L, introducing air at 35-55 DEG C until dissolved oxygen saturation in order to oxidize glucose into gluconic acid, and controlling the pH value to 5.5-8.0 to convert gluconic acid into sodium gluconate; and (4) refining: refining the sodium gluconate obtained in step (3) in order to obtain the finished sodium gluconate. Themethod adopts a cellulase preparation for catalyzing the saccharification of lignocellulose, so the problems limited sodium gluconate raw material, complicated process and low yield are solved.

Description

technical field

[0001] The invention belongs to the technical field of biochemical industry, and in particular relates to a method for preparing sodium gluconate from lignocellulose raw materials. Background technique

[0002] Sodium gluconate is a sodium salt of gluconic acid, a deep-processed product of glucose, and a basic raw material for gluconolactone, gluconate (zinc, copper, ferrous salt), etc. At present, the production methods of sodium gluconate mainly include four methods: biological fermentation and enzyme catalysis, homogeneous chemical oxidation, electrolytic oxidation and heterogeneous catalytic oxidation; in industrial production, heterogeneous catalytic oxidation and microbial fermentation law. Among them, the heterogeneous catalytic oxidation method uses rare metals as the main catalyst, which is expensive, which limits the application range and development space of this method. Microbial fermentation has developed rapidly in recent years and has become ...

Examples

Embodiment 1

[0046] Embodiment 1: By the method of indirect connection, construct the cellulase preparation based on Clostridium thermocellum cellulite

[0047] Through seamless cloning, the tdk expression cassette (including the promoter of the gapDH gene) and the pyrF expression cassette (including 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.

[0048] The cellulase Cel9K (exocellulase, encoded by the nucleic acid sequence 2113813 to 2111293 in the genome CP002416.1) in the celluloso...

Embodiment 2

[0054] Embodiment 2: By the method of indirect connection, construct the cellulase preparation based on Clostridium thermocellum cellulite

[0055] The difference from Example 1 is that the polypeptide fragment II or polypeptide fragment I is linked to the 3' end of cellulosome endonuclease CelZ (SEQ ID NO: 15). The constructed recombinant strain is cultured to the mid-logarithmic phase in GS-2 medium with 5 grams per liter of cellulose or cellobiose as the carbon source, and can be used as a whole-bacteria enzyme preparation for biosaccharification of lignocellulose.

Embodiment 3

[0056] Embodiment 3: By the method of direct connection, construct the cellulase preparation based on Clostridium thermocellum cellulosome

[0057] Using the overlap extension polymerase chain reaction method, cellulosic exonuclease Cel9-48 (SEQ ID NO: 16) was combined with the sequence of type II adhesion module CohIIct (SEQ ID NO: 7) or type I of Clostridium thermocellum The sequence of the docking module DocIct (SEQID NO:6) was directly connected, wherein the sequence of CohIIct or DocIct was connected to the 3' end of the Cel9-48 sequence to obtain the sequence of Cel9-48-DocIct or Cel9-48-CohIIct.

[0058] Using the Cel9-48-DocIct or Cel9-48-CohIIct sequence as the target sequence, using the MluI and EagI restriction sites to clone into the homologous recombination plasmid pHK-HR, using the lactate dehydrogenase gene clo1313_1160 as the targeted replacement sequence, The homologous recombination plasmid pHK-HR-cel9-48 was constructed. The upstream homology arm HR-up is t...

technical field

[0001] The invention belongs to the technical field of biochemical industry, and in particular relates to a method for preparing sodium gluconate from lignocellulose raw materials. Background technique

[0002] Sodium gluconate is a sodium salt of gluconic acid, a deep-processed product of glucose, and a basic raw material for gluconolactone, gluconate (zinc, copper, ferrous salt), etc. At present, the production methods of sodium gluconate mainly include four methods: biological fermentation and enzyme catalysis, homogeneous chemical oxidation, electrolytic oxidation and heterogeneous catalytic oxidation; in industrial production, heterogeneous catalytic oxidation and microbial fermentation law. Among them, the heterogeneous catalytic oxidation method uses rare metals as the main catalyst, which is expensive, which limits the application range and development space of this method. Microbial fermentation has developed rapidly in recent years and has become ...

Examples

Embodiment 1

[0046] Embodiment 1: By the method of indirect connection, construct the cellulase preparation based on Clostridium thermocellum cellulite

[0047] Through seamless cloning, the tdk expression cassette (including the promoter of the gapDH gene) and the pyrF expression cassette (including 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.

[0048] The cellulase Cel9K (exocellulase, encoded by the nucleic acid sequence 2113813 to 2111293 in the genome CP002416.1) in the celluloso...

Embodiment 2

[0054] Embodiment 2: By the method of indirect connection, construct the cellulase preparation based on Clostridium thermocellum cellulite

[0055] The difference from Example 1 is that the polypeptide fragment II or polypeptide fragment I is linked to the 3' end of cellulosome endonuclease CelZ (SEQ ID NO: 15). The constructed recombinant strain is cultured to the mid-logarithmic phase in GS-2 medium with 5 grams per liter of cellulose or cellobiose as the carbon source, and can be used as a whole-bacteria enzyme preparation for biosaccharification of lignocellulose.

Embodiment 3

[0056] Embodiment 3: By the method of direct connection, construct the cellulase preparation based on Clostridium thermocellum cellulosome

[0057] Using the overlap extension polymerase chain reaction method, cellulosic exonuclease Cel9-48 (SEQ ID NO: 16) was combined with the sequence of type II adhesion module CohIIct (SEQ ID NO: 7) or type I of Clostridium thermocellum The sequence of the docking module DocIct (SEQID NO:6) was directly connected, wherein the sequence of CohIIct or DocIct was connected to the 3' end of the Cel9-48 sequence to obtain the sequence of Cel9-48-DocIct or Cel9-48-CohIIct.

[0058] Using the Cel9-48-DocIct or Cel9-48-CohIIct sequence as the target sequence, using the MluI and EagI restriction sites to clone into the homologous recombination plasmid pHK-HR, using the lactate dehydrogenase gene clo1313_1160 as the targeted replacement sequence, The homologous recombination plasmid pHK-HR-cel9-48 was constructed. The upstream homology arm HR-up is t...

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