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Novel microbial enzymes and their use

一种引物对、酪氨酸酶的技术，应用在新的真菌酶蛋白及其应用领域

Inactive Publication Date: 2008-03-05
VALTION TEKNILLINEN TUTKIMUSKESKUS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These enzymes are suitable for protein modification

Method used

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  • Novel microbial enzymes and their use
  • Novel microbial enzymes and their use
  • Novel microbial enzymes and their use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Plate screening of tyrosinase positive microorganisms

[0070] Indicators for tyrosinase activity screening were selected according to the literature. L-tyrosine, p-cresol, p-coumaric acid, tyramine, 3-hydroxy-2-aminobenzoic acid, and catechin were used at the concentrations indicated in Table 1. Trichoderma reesei was grown on wort agar containing selected indicators (Table 1) (Difco) for 48 days at 37°C. Visually inspect the plate for possible color changes.

[0071] T. reesei showed clear positive reactions with L-tyrosine, tyramine, 3-hydroxy-2-aminobenzoic acid and catechins. The results clearly showed that T. reesei was a tyrosinase-positive microorganism.

[0072] Table 1. Plate test screening of tyrosinase activity from T. reesei

[0073] indicator

Embodiment 2

[0075] Isolation of tyr1 and tyr2 genes from Trichoderma reesei

[0076] Two novel tyrosinase genes were amplified from genomic T. reesei DNA by PCR. Primers for tyr1 are forward:

[0077] GCT ACC GCG GAT GGG CTT CCT CGC TCG CCT CAC (SEQ ID NO: 5)

[0078] and the reverse:

[0079] CTG AGG ATC CTC AGT GGT GGT GGT GGT GGT GCT CCC ACA ACA CCAATC TCA GCA T (SEQ ID NO: 6).

[0080] Use forward primer:

[0081] GGG GAC AAG TTT GTA CAA AAA AGC AGG CTA TCA TGC TGT TGT CAGGTC CCT CTC G (SEQ ID NO: 7) and reverse primer:

[0082] GGG GAC CAC TTT GTA CAA GAA AGC TGG GTC AGT GGT GGT GGT GGTGGT GCA GAG GAG GGA TAT GGG GAA CGG CAA A (SEQ ID NO: 8) amplifies the tyr2 gene.

[0083]PCR reactions were performed with Dynazyme EXT thermostable polymerase (Finnzymes, Finland) in the reaction mixture recommended by the manufacturer. The PCR program had an initial denaturation step of 3 min at 94 °C, followed by 25 cycles of 30 s at 94 °C, 45 s at 52 °C and 2.5 min at 72 °C. This was follow...

Embodiment 3

[0085] Overexpression of the tyr2 gene in Trichoderma reesei under a strong promoter

[0086] The tyr2 gene fragment was transferred from pDONR221 vector to T. reesei expression vector pMS186 by LR recombination reaction. This vector contains the Gateway reading frame cassette C (RfC) inserted between the cbhl (cellobiohydrolase 1) promoter and terminator. This vector also has a hygromycin resistance cassette for selection of T. reesei transformants. LR recombination reactions were performed using the Gateway recombination kit (Invitrogen) as directed by the manufacturer. During this recombination process, the tyr2 gene fragment was inserted between the cbh1 promoter and terminator, thereby obtaining plasmid pMS190 (Fig. 2).

[0087] Plasmid pMS190 was transformed into T. reesei strain VTT-D-00775 essentially as described (Penttil et al., 1987) and hygromycin resistant transformants were selected on plates containing 125 μg / ml hygromycin B. Transformants were streaked three...

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Abstract

The invention relates to extracellular tyrosinases obtainable from Trichoderma spp. and to methods and means for producing them by recombinant technology. The enzymes are particularly useful in cross-linking food proteins.

Description

field of invention [0001] The present invention relates to enzyme technology, and more particularly to novel fungal enzyme proteins and their use. The present invention further relates to polynucleotides encoding said enzymes, methods of producing them and expression vectors and host cells for producing said enzymes. Background of the invention [0002] Enzymes are used in various types of production processes in the pulp and paper industry, the textile industry, and the food, feed, and beverage industries. Enzymes are also used in the cosmetic and pharmaceutical industries and in detergents. Industrial enzymes may be of animal, vegetable or microbial origin and are preferably extracellular enzymes. They are generally more stable and readily produced by recombinant techniques. Isolation and purification of intracellular enzymes from host cells is costly and laborious and it is therefore advantageous if said enzymes are extracellular enzymes, ie proteins secreted from the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/02C12N15/53C12N15/80D06M16/00C12S3/00A23J3/00A23J3/34A23L7/10C12ND06M15/15
CPCD06M16/003A23J3/34D06M15/15C12N9/0059D06M2101/12A23J3/00C12N9/0004C12N15/52
Inventor K·克鲁斯E·塞林海莫K·奥蒂奥J·布克特M·萨洛黑莫R·兰托
Owner VALTION TEKNILLINEN TUTKIMUSKESKUS
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