Mutant beta-glucosidase variants with increased thermostability

一种氨基酸、序列的技术，应用在糖基化酶、酶、水解酶等方向，能够解决不溶性纤维素不是等问题，达到防止终产物抑制作用、增加酶活性、改良热稳定性的效果

Inactive Publication Date: 2018-08-03
TECH UNIV MUNCHEN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, insoluble cellulose is not a homogeneous crystal

Method used

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  • Mutant beta-glucosidase variants with increased thermostability
  • Mutant beta-glucosidase variants with increased thermostability
  • Mutant beta-glucosidase variants with increased thermostability

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0240] Example 1: Isolation of the cglT wild-type gene

[0241] The complete gene cglT from Thermoanaerobe brucei was synthesized in codon usage in E. coli and cloned into the pET24a(+) expression vector (Novagen, Germany) fused to a C-terminal His-tag. Escherichia coli DH10B cell (Invitrogen, USA) plasmid DNA was isolated and transformed into Escherichia coli BL21Star TM (DE3) cells (Invitrogen, USA) were used for recombinant protein expression. Cells were grown in LB medium containing 100 μg / ml ampicillin (w / v) and incubated at 37°C. Liquid cultures (same medium) were shaken in a rotary shaker at 180 rpm at 37°C. LB medium contains 5 g of yeast extract, 10 g of tryptone, and 10 g of NaCl / L double distilled water; add NaOH to adjust the pH to 7.2. 16 g / l agar was added to solidify the medium.

Embodiment 2

[0242] Example 2: Mutagenesis of the cglT wild-type gene

[0243] Point mutations in the wild-type cglT gene leading to potentially stabilizing amino acid exchanges were introduced in the wild-type cglT gene by PCR site-directed mutagenesis using synthetic oligonucleotide pairs with appropriate mismatches (Table 1).

[0244] Table 1: Mutagenesis primers used

[0245] Primer

Sequence (5`→3`)

mutation

SEQ ID NO:

V111K_neu

AGCGCGATATTAAACCCGCAGCGACCATTTATC

V111K

45

V111K_rev_neu

GATAAATGGTCGCTGCGGGTTTAATATCGCGCT

46

V293I

CGATTGACTTCTTAGGCATCAATTACTACACTC

V293I

47

V293I_rev

GAGTGTAGTAATTGATGCCTAAGAAGTCAATCG

48

T423E

ATTGTGTATGTGGACTATGAGACCCAGAAACG

T423E

49

T423E_rev

CGTTTCTGGGTCTCATAGTCCACATACACAAT

50

L441R

ACAAAGAGGTGATTCGCGATGATGGGATTGAAG

L441R

51

L441R_rev

CTTCAATCCCATCATCGCGAATCACCCTTTTTGT

52

[0246] Due to ...

Embodiment 3

[0248] Example 3: Recombinant Expression of Mutant Polypeptides

[0249] Transformation of plasmids carrying wild-type and mutant β-glucosidase genes into chemically competent Escherichia coli BL21Star TM (DE3) cells for protein expression. Precultures were prepared from single colonies in liquid LB medium. After 6-8 hours of growth under aeration, expression cultures were prepared by inoculating ZYP 5052 auto-induction medium containing 2 g / l lactose and the cultures were incubated overnight [Studier, F.W., Protein production by auto-induction in high density shaking cultures . Protein Expr Purif, 2005.41:207-34.2]. Cells were harvested by centrifugation (4500 rpm, 10 min, 4°C) and cell pellets were frozen at -20°C until further use. To lyse cells, thaw cell pellets on ice, resuspend in cell lysis buffer (50mM MOPS pH 7.3, 0.5M NaCl, 20mM imidazole, 20mM CaCl 2 ) to which protease inhibitor cocktail (cOmplete, Mini, EDTA-free; pH 7.4; Hoffmann-La Roche AG, Switzerland) and ...

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Abstract

The invention relates to mutant variants of the beta-glucosidase Cgl T from Thermoanaerobacter brockii and nucleic acids for producing the same. Said mutant variants show significantly increased thermostability and enzyme activity. Furthermore, the invention provides vectors, host cells and methods for producing said mutant variants of the beta-glucosidase Cgl T. Also provided are artificial cellulosomes comprising the mutant variants of the beta-glucosidase Cgl T and methods for the enzymatic hydrolysis of cellulosic biomass comprising said artificial cellulosomes and / or said mutant variantsof the beta-glucosidase Cgl T.

Description

field of invention [0001] The present invention relates to mutant variants of the beta-glucosidase CglT from Thermoanaerobacter brockii and nucleic acids producing them. The mutant variants showed significantly increased thermostability and enzymatic activity. The present invention further provides vectors, host cells and methods for producing said mutant variants of β-glucosidase CglT. The present invention also provides artificial cellulosomes comprising mutants of β-glucosidase CglT and enzymatic hydrolysis of mutants comprising said artificial cellulosomes and / or said β-glucosidase CglT. A method for the production of cellulosic biomass. Background of the invention [0002] Biomass contains a variety of polysaccharides as structural or storage compounds. Cellulose and hemicellulose are the most important. Availability and ease of use make biomass an abundant renewable energy source. Approximately half of the carbon-containing compounds in terrestrial biomass are cel...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/42
CPCC12Y302/01021C12N9/2445C12N15/86C12P19/14
Inventor V·兹韦尔洛夫W·施瓦茨R·普雷希特尔B·莱斯C·黑尔德W·利布尔
Owner TECH UNIV MUNCHEN
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