Methods for producing lipases
By changing the polynucleotide sequence of lipase to introduce disulfide bonds, reducing the hydrolysis activity and reactivating it under the action of an activator, the problem of insufficient stability and washing performance of existing lipase in harsh environments is solved, achieving a more efficient Lipid stain removal.
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[0183] Preparation of variants
[0184] The present invention also relates to methods for obtaining lipase variants and / or fragments thereof, these methods comprising introducing into the parent lipase at positions -57, -54, -47, -46,-corresponding to SEQ ID NO: 2 One of 43, -40, -38, -37, -36, -33, -31, -24, 101, 102, 104, 120, 182, 186, 212, 216, 239, 240, 246, and 249 or Substitution at multiple positions, where the variant and / or fragment has lipase activity; and recovering the variant and / or fragment.
[0185] The present invention also relates to methods for obtaining lipase variants and / or fragments thereof. These methods include introducing into the parent lipase at positions corresponding to -57, -54, -47, -46, -43, -40,- 38, -37, -36, -33, -31, and -24 substitutions at one or more positions, and included in positions 101, 102, 104, 120, 182, 186 corresponding to SEQ ID NO: 2 , 212, 216, 239, 240, 246, and 249 at one or more positions.
[0186] In some aspects, the presen...
example
[0614] The following examples include Rhizomucor miehei lipase (RmL) and Thermomyces lanuginosa lipase (TlL) variants. Two such preferred RmL variants are called RPPI0018 (P182C T-40C) and RPPI0019 (N186C P-46C).
[0615] SEQ ID No: 1 lists the nucleotide sequence of RmL, including the sequence encoding the following cleaved prepropeptide (underlined):
[0616]
[0617]
[0618] SEQ ID No: 2 lists the amino acid sequence of RmL, including the following cleaved preprosequence (underlined):
[0619]
[0620] SEQ ID No: 3 lists the amino acid sequence of the RmL mature protein:
[0621]
[0622]
[0623] SEQ ID No: 4 lists the amino acid sequence of the propeptide of RmL:
[0624] VPIKRQSNST VDSLPPLIPS RTSAPSSSPS TTDPEAPAMS RNGPLPSDVE TKYGMALNATSYPDSVVQAM
[0625] SEQ ID No: 5 lists the nucleotide sequence of TlL, including the sequence encoding the following cleaved prepropeptide (underlined):
[0626]
[0627] SEQ ID No: 6 lists the amino acid sequence of TlL, including the following clea...
example 1
[0639] Example 1: Thermal stability
[0640] Using a VP-Capillary Differential Scanning Calorimeter (MicroCal Inc., Piscataway, New Jersey, USA), the RmL wild type together with the RmL variant was determined by differential scanning calorimetry (DSC) The thermal stability of RPPI0018 and RPPI0019. At a constant programmed heating rate of 200K / hr, heating in buffer (50mM HEPES; pH 8.0; 0.2mM CaCl 2 In the thermogram (Cp vs. T) obtained after the enzyme solution in ), the thermal denaturation temperature Td (°C) is regarded as the top of the denaturation peak (main endothermic peak). A similar experiment was run in which 1 mM LAS (anionic surfactant) or 0.05 mg / mL Relase (thermostable protease, Novozymes) was added to the buffer. Load the sample solution and reference solution (approximately 0.2 mL) into the calorimeter (reference solution: buffer without enzyme) from the storage condition of 10°C, and before the DSC scan from 20°C to 100°C, Heat pre-equilibration at 20°C for ab...
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