Endoglucanase variants with increased activity and uses thereof
A technology of endoglucanase and uses, which is applied to the endoglucanase variant with improved activity and the field of use thereof, and can solve problems such as cellulase reduction and the like
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Embodiment 1
[0090] Example 1: Evolution via L-shuffling
[0091] The gene sequence of Trichoderma reesei endoglucanase 2 (EG2) was identified with the putative Botrytis cinerea endoglucanase 2 genes ( BF gene) and the putative S. sclerotiorum endoglucanase 2 gene (SS gene) were subjected to one round of L-shuffling according to the patented method described in EP1104457B11.
[0092] 1- High Throughput Screening
[0093] A high-throughput screening assay was developed to select the best clones produced by L-shuffling, eg, those showing at least a 20% increase in endoglucanase activity relative to the enzyme from Trichoderma reesei.
[0094] High-throughput screening assays are performed according to the following steps:
[0095] - isolation of E. coli clones expressing a variant of the recombinase according to the invention on agar and pre-culturing the clones in LB medium overnight at 37°C;
[0096] - Inoculate 6% LB medium with the preculture, then incubate at 37°C for 5 hours, then...
Embodiment 2
[0152] Variants 146C4, 191H11, 222E1 and 225C7, as well as the EG2 reference gene of Trichoderma reesei (SEQ ID NO: 2) were cloned into cbh1 of the pUT1040 plasmid containing the phleomycin resistance gene as marker by BamH1 / XhoI double digestion between the promoter and terminator. Trichoderma reesei strain CL847ΔEG1 was transformed with 5 μg of each vector. Protoplasts were transformed with 5 μg of each construct by calcium and PEG shock following conventional methods known to those skilled in the art. Transformants were selected on PDA / sucrose selection medium containing 30 μg / l phleomycin. After three consecutive subcultures where pure clones could be obtained, 11-15 clones were obtained for each variant. in F45 medium (800 μl 85% H 3 PO 4 , 4.2g (NH 4 ) 2 SO 4 , 0.3g MgSO 4 .7H 2 All clones were grown in O, 0.75 g corn steep liquor, 1 ml Oligo Ferment, 6 g potassium phthalate, pH 5.8-6). After 7 days of incubation at 30°C, the supernatant was removed and 10 mg / l...
Embodiment 3
[0158] Example 3: Recombinant expression of EG2 reference endoglucanase and 222E1 and 225C7 variants in Saccharomyces cerevisiae
[0159] 1- Production of EG2 reference endoglucanase reference protein and its 222E1 variant in extracellular medium
[0160] The EG2 reference endoglumerase gene of Trichoderma reesei (SEQ ID NO: 1) and the genes of the 222E1 and 225C7 variants (SEQ ID NO: 23 and 25, respectively) were cloned in the absence of their signal peptides into the pESC-LeuαΔmyc vector (CNRS-CERMAV). This construct allows protein expression in the medium of Saccharomyces cerevisiae strain EBY100, which is auxotrophic for leucine and tryptophan (Boder ET and Wittrup KD, Biotechnol Prog, 1998, 14:55-62). This plasmid is capable of placing gene expression under the control of the galactose-inducible GAL1 promoter and has an auxotrophic selectable marker gene (Leu2) allowing selection of transformants.
[0161] Transformation of Saccharomyces cerevisiae EBY100 was performe...
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