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Method for preparing squalene by using lignocellulose

A technology of lignocellulose and squalene, applied in chemical instruments and methods, purification/separation of hydrocarbons, extraction purification/separation, etc., can solve the problem that the conversion efficiency cannot meet the requirements of industrial production, increase the amount of enzyme addition or the number of additions, Can not interact with cellulite and other problems, to achieve the effect of solving the problem of comprehensive utilization, the use of water-saving chemicals, and the source of raw materials is cheap

Active Publication Date: 2021-04-27
青岛中科潮生生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, since the activity and stability of exogenously added proteins will decrease with the progress of the saccharification process, the consumption of enzymes requires an increase in the amount or frequency of enzyme addition
In addition, these non-cellulosomal proteins are added to the saccharification system as free enzymes, which cannot interact with cellulosomes, and will significantly reduce the synergistic effect with other enzymes in the system, inevitably resulting in additional enzymes that are greater than the demand, resulting in The high cost of enzyme preparations
Not only that, this saccharification strategy that relies on the addition of exogenous free enzymes has complex processes, high equipment requirements, and conversion efficiency that cannot meet the requirements of industrial production.

Method used

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  • Method for preparing squalene by using lignocellulose
  • Method for preparing squalene by using lignocellulose

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Embodiment 1: By the method of indirect connection, construct the cellulase preparation based on Clostridium thermocellum cellulite

[0049] 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.

[0050] The cellulase Cel9K (exocellulase, encoded by the nucleic acid sequence 2113813 to 2111293 in the genome CP002416.1) in the celluloso...

Embodiment 2

[0056] Embodiment 2: By the method of indirect connection, construct the cellulase preparation based on Clostridium thermocellum cellulite

[0057] 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

[0058] Embodiment 3: By the method of direct connection, construct the cellulase preparation based on Clostridium thermocellum cellulosome

[0059] 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 (SEQ ID 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.

[0060] 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...

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Abstract

Aiming at the problems existing in the production of squalene by microbial fermentation in the prior art, the invention provides a process for preparing squalene by using lignocellulosic agricultural and forestry wastes as raw materials. The process includes the steps of pretreatment, saccharification, yeast-like fermentation, squalene extraction and the like. The process adopts the combination of lignocellulosic biosaccharification and yeast-like high-density fermentation; by using lignocellulosic biomass as raw material, on the one hand, the cost of enzymes used in the saccharification stage and the production cost of squalene microbial fermentation are significantly reduced On the other hand, it also solves the problem of comprehensive utilization of agricultural and forestry waste. In addition, the lignocellulose saccharification stage medium and fermentation medium can be recycled, which can significantly save water and reduce the use of chemicals, and has a significant effect of reducing waste water discharge and reducing costs. In the process of the invention, the squalene output can reach 13g / L, accounting for 5-9% of the dry weight of the bacteria, and the purity of the refined squalene is greater than 93%.

Description

technical field [0001] The invention relates to the field of microbial fermentation engineering, in particular to a method for producing squalene by using lignocellulose raw materials. Background technique [0002] Squalene is a non-toxic marine biologically active substance with the effect of preventing and curing diseases. Its chemical name is 2,6,10,15,19,23-hexamethyl-2,6,10,14 ,18,22-Tetracosahexaene has many physiological functions such as increasing the activity of superoxide dismutase in the body, enhancing the body's immunity, improving sexual function, anti-aging, anti-fatigue, anti-tumor, etc. It is widely used in medicine, Beauty, cosmetics, food and other fields. [0003] Currently, the main source of squalene is fish oil, especially shark liver oil. However, with the depletion of fishery resources and the improvement of relevant regulations, people are trying to find other sources of squalene; such as from olive oil, palm oil and other oil crops, or from amar...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P5/02C12P19/14C12P19/02C07C7/00C07C7/04C07C7/10C07C11/21
CPCC07C7/005C07C7/04C07C7/10C12P5/026C12P19/02C12P19/14C07C11/21Y02E50/30
Inventor 崔球刘亚君宋晓金
Owner 青岛中科潮生生物技术有限公司
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