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Publication Number
US-11655463-B2
Publication Date
2023-05-23
Expiration Date
Abstract
A modified endoinulinase is provided, comprising modified wild-type T. purpuregenus endoinulinase, or a functional fragment thereof, in which an amino acid residue at each one of one or more positions corresponding to 128, 316, 344, 350 or 504 of wild-type T. purpuregenus endoinulinase is substituted, wherein: (i) a tyrosine residue corresponding to Y128 is substituted with H, K or R; a glutamate residue corresponding to E344 is substituted with K, H or R; and a threonine residue corresponding to T504 is substituted with M, S or Y; and optionally an alanine residue corresponding to A316 is substituted with T, S, C or M; (ii) a tyrosine residue corresponding to Y128 is substituted with H, K or R; a glutamate residue corresponding to E344 is substituted with K, H or R; a threonine residue corresponding to T504 is substituted with M, S or Y; and a glutamine residue corresponding to Q350 is substituted with L, G, A, V or I; or (iii) a tyrosine residue corresponding to Y128 is substituted with H, K or R.
Core Innovation
The disclosed invention relates to improved modified wild-type Talaromyces purpureogenus endoinulinases. The modified endoinulinase comprises an amino acid sequence set forth in SEQ ID NO: 1, with substitutions defined at positions corresponding to Y128, E344, and T504, and optionally additional substitutions at positions corresponding to A316 and/or Q350.
The invention addresses performance limitations of recombinant T. purpureogenus wild-type endoinulinase by providing modified variants characterized by improved functional activity or solubility compared to the wild-type endoinulinase. Reported functional improvements include higher soluble expression, increased hydrolysis activity in cell lysates versus recombinant wild type, and a shifted pH optimum to above pH 5.
The disclosure further includes functional fragments of the modified endoinulinase having at least 90% identity to a T. purpureogenus wild-type sequence. The modified variants also show altered product distribution for fructooligosaccharides and enhanced dependence on metal ions, notably Mg2+.
Claims Coverage
The independent claim covers a modified wild-type T. purpureogenus endoinulinase with specific substitutions at positions corresponding to E344 and/or T504, optionally combined with additional substitutions at positions corresponding to 128, 316, or 350, and characterized by improved functional activity or solubility compared to the wild-type endoinulinase. The core inventive coverage includes three inventive features.
Modified wild-type T. purpureogenus endoinulinase defined by E344 and/or T504 substitutions
A modified wild-type T. purpureogenus endoinulinase comprising an amino acid sequence set forth in SEQ ID NO: 1 with a substitution being any one of: E344 to K, H, or R; T504 to M, S, or Y; or both, or a functional fragment thereof comprising at least 90% identity to a T. purpureogenus wild-type endoinulinase sequence.
Optional additional substitutions at positions corresponding to 128, 316, or 350
The functional fragment or modified endoinulinase further comprises one or more amino acid substitutions in positions corresponding to position 128, 316, or 350 of the wild-type T. purpureogenus endoinulinase.
Characterization by improved functional activity or solubility compared to wild type
The modified endoinulinase is characterized by improved functional activity or solubility compared to the wild-type T. purpureogenus endoinulinase.
The claims center on a modified T. purpureogenus endoinulinase defined by E344 and/or T504 substitutions, optional additional substitutions at 128, 316, and/or 350, and a requirement for improved functional activity or solubility relative to the wild-type enzyme.
Stated Advantages
Improved functional activity compared to the wild-type Talaromyces purpureogenus endoinulinase.
Improved solubility compared to the wild-type Talaromyces purpureogenus endoinulinase.
Higher soluble expression.
Increased hydrolysis activity in cell lysates versus recombinant wild type.
Shifted pH optimum to above pH 5.
Altered product distribution for fructooligosaccharides.
Enhanced dependence on metal ions, notably Mg2+.
Documented Applications
Producing fructooligosaccharides by contacting inulin with a modified endoinulinase or a functional fragment of it.
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