Protein hydrolysates with increased yield of n-terminal amino acid
A kind of technology of protein hydrolysis and proteolytic enzyme, applied in the field of hydrolysate of free amino acid yield
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[0141] Preparation of Trichoderma sp. for transformation may, for example, involve the preparation of protoplasts from fungal mycelium. See Campbell et al. (1989) Curr. Genet. 16:53-56. Mycelia can be obtained from germinated vegetative spores. Protoplasts are produced by treating the mycelia with enzymes that digest the cell walls. Protoplasts are protected by the presence of osmotic stabilizers in the suspension medium. These stabilizers include sorbitol, mannitol, potassium chloride, magnesium sulfate, and the like. Usually the concentration of these stabilizers varies between 0.8M and 1.2M, for example a 1.2M solution of sorbitol can be used in the suspension medium.
[0142] DNA uptake into the host Trichoderma sp. strain depends on the calcium ion concentration. Typically, between about 10 mM and 50 mM CaCl is used in the uptake solution 2 . Additional suitable compounds include buffer systems such as TE buffer (10 mM Tris, pH 7.4; 1 mM EDTA) or 10 mM MOPS (pH 6.0)...
example 1
[0176] Cloning of Example 1 Fungal X-Pro Protease
[0177] Two fungal strains, Melanocarpus albomyces CBS 177.67 (GICC #2522192) and Malbranchea cinamonea CBS 343.55 (GICC #2518670), were selected as potential candidates for enzymes useful in a variety of industrial applications. source. Thermoalcanthus CBS177.67 and C. camphora CBS 343.55 were purchased from the CBS-KNAW Fungal Biodiversity Center (Uppsalalaan 8, 3584CT Utrecht, The Netherlands). Chromosomal DNA was sequenced using Illumina's next-generation sequencing technology, and two fungal X-Pro proteases were identified after annotation: MalPro11 from C. pyrromyces CBS177.67, and MciPro4 from C. camphoria CBS 343.55. The full-length protein sequences of MalPro11 and MciPro4 are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.
[0178]Three fungal strains listed in the JGI database (https: / / genome.jgi.doe.gov / portal / ) (Trichoderma citrinoviride TUCIM 6016, Fusarium verticillioides 7600 and Stagonospora sp. SRC11...
example 2
[0179] Expression of the identified fungal X-Pro protease of example 2
[0180] DNA sequences encoding full-length MalPro11, MciPro4 or TciPro1 (after an additional 5' DNA fragment (SEQ ID NO: 6)) were chemically synthesized and inserted into T. reesei expression vector pGXT (as published in PCT application WO2015 pTTTpyr2 vector described in / 017256, which application is incorporated herein by reference). The resulting plasmids were labeled pGXT-MalPro11, pGXT-MciPro4 and pGXT-TciPro1. Using protoplast transformation (Te'o et al. (2002) J. Microbiol. Methods [Journal of Microbial Methods] 51:393-99) each individual expression vector was then transformed into a suitable Trichoderma reesei strain (described in published PCT application WO 05 / 001036). Transformants were selected on media containing acetamide as the sole nitrogen source. After 5 days of growth on acetamide plates, transformants were harvested and fermented in defined media containing a mixture of glucose and s...
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