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Artificial cellulosome and the use of the same for enzymatic breakdown of resilient substrates

A technology of complexes and derivatives, applied in the direction of enzymes, enzymes, enzyme stabilization, etc., can solve the problems that separation cannot be easily separated, inefficient, and expensive for industrial development.

Inactive Publication Date: 2013-04-03
TECH UNIV MUNCHEN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these attempts have so far failed due to insurmountable difficulties in separating the components in their native state - the tight associations in the complex do not allow easy separation using mild, non-denaturing methods
[0025] Enzymatic breakdown of insoluble crystalline substances such as crystalline cellulose and heterogeneous hemicellulose remains inefficient and slow and requires high enzyme concentrations, making industrial development expensive and relatively inefficient using today's enzyme preparations

Method used

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  • Artificial cellulosome and the use of the same for enzymatic breakdown of resilient substrates
  • Artificial cellulosome and the use of the same for enzymatic breakdown of resilient substrates
  • Artificial cellulosome and the use of the same for enzymatic breakdown of resilient substrates

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0108] Example 1: Isolation of mutants that do not form cellulosomes

[0109] Mutants were isolated from mutagenized cultures of C. thermocellulosa (Figure 1). Six colonies with decreased or lost ability to form a clear color circle in the cellulose surrounding the colony were randomly selected. One of the C. thermocellum mutants, SM1, completely lost the ability to produce the scaffold protein CipA or active adhesion factors. Enzymes from wild type (with cellulosomes) and from mutant SM1 (without cellulosomes; free enzyme). Enzyme activity on barley β-glucan and CMC was about 8.5 and 1.0 U mg for the two strains, respectively -1 Protein (Table 1). In contrast, in the mutant SM1, the specific activity on crystalline cellulose dropped dramatically, up to 15-fold compared to the wild type.

[0110] Except for the complete disappearance of the CipA component (scaffold protein CipA), the mutant produced roughly the same amount of cellulosomal components as compared to the w...

Embodiment 2

[0113] Example 2: Reconstitution of cellulosomes

[0114] A: Preparation of Enzyme Components

[0115] Mutant SM1 and mutant supernatant protein (SM901) were selected to reconstitute artificial cellulosomes. In addition, genes encoding cellulase components were cloned and characterized for their activity on biochemical parameters such as pH and temperature optima, as well as on different substrates. From previous data on cellulosome composition 11 Five of the most prominent enzyme components with cellulase activity were selected. Additionally, biochemical characterization of β-glucosidases from a large number of thermophilic saccharolytic bacteria. The β-glucosidase BglB from Thermotoga maritima was selected for its high thermostability and high activity on cellodextrins. This gene was fused to the dockerin module of the C. thermocellulase cellulase CelA. Determine optimal expression conditions. CelK-d1, CelR-d1, CelT-d1, CelE-d1, CelS-d1 and BglB-d1 are referred to ...

Embodiment 3

[0125] Example 3: Activity of artificial cellulosome complexes

[0126] This mixture of complexes with and without CBM is now bound on the surface of polystyrene nanoparticles via optimized aliphatic linker molecules. This structure is schematically shown in FIG. 3 . Despite the inevitable loss of steric hindrance and freedom of enzymatic components due to the dense coverage of nanoparticles, the incorporation of various mini-scaffold protein complexes also resulted in increased activity during hydrolysis of crystalline cellulose (Fig. 3, 4). Additionally, the pH range of the enzyme is wider if the protein is bound to the particle (Figure 5). This also holds true for the temperature stability of the cellulase (Figure 6). Both results are important advantages for technical applications.

[0127] To test the feasibility of this approach, a portion of the SM901 component mixture was replaced with one or more recombinant cellulases. Although the SM901 component in the mixtur...

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Abstract

The present invention relates to an in vitro produced, artificial cellulosome for enzymatic breakdown of resilient substrates. In particular, the present invention provides a complex having an increased activity on resilient substrates, such as crystalline cellulose. The in vitro formed complex comprises a backbone scaffold having at least four binding sites capable of binding the enzyme components, whereby at least two of the binding sites have essentially the same binding specificity; and at least three different enzyme components being randomly bound to the at least four binding sites. Method for preparing the complex and uses of the same for enzymatic breakdown of resilient substrates are also provided.

Description

technical field [0001] The present invention provides an artificial cellulosome for enzymatically decomposing resilient substrates. In particular, the present invention provides a complex comprising: a backbone scaffold having at least four binding sites capable of binding enzyme components, wherein at least two of the binding sites have essentially and at least three different enzyme components randomly binding to said at least four binding sites. In addition, the invention relates to a method for the preparation of said complex. Furthermore, the invention relates to the use of said complexes and said different enzyme components for the enzymatic breakdown of elastic substrates such as cellulose. Background technique [0002] Cellulose is an abundant renewable energy source for biotechnology, the production of biofuels and a building block for chemicals. With the imminent advent of second-generation white biotechnology, cellulose from lignocellulosic biomass will become ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P7/10C12N9/42
CPCC12Y302/01006Y02E50/16C07K2319/20C12Y302/01021C12N9/244C12Y302/01004C12P19/14C12N9/2434C12N9/2445C12N9/96C12N9/2437Y02E50/10
Inventor 沃尔夫冈·H·施瓦茨扬·克劳斯弗拉迪米尔·V·茲维勒夫丹尼尔·霍恩布尔格达尼埃拉·柯克路易斯-菲利普·许尔德
Owner TECH UNIV MUNCHEN
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