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Publication Number
US-10941386-B2
Publication Date
2021-03-09
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 invention relates to a modified endoinulinase derived from T. purpuregenus wild-type endoinulinase. The modified endoinulinase comprises an amino acid sequence set forth in SEQ ID NO: 1, with a substitution of tyrosine at position 128 (Y128) to H, K, or R, or a functional fragment thereof. The modified endoinulinase or functional fragment thereof further comprises an amino acid sequence having at least 90% identity to a T. purpuregenus wild-type sequence of endoinulinase set forth in SEQ ID NO: 3.
The modified endoinulinase optionally further comprises one or more amino acid substitutions in positions corresponding to position 316, 344, 350 or 504 of the wild-type T. purpuregenus endoinulinase. In particular embodiments, substitutions include E344 to K, H, or R, T504 to M, S, or Y, and optional substitutions at A316 to T, S, C, or M and/or Q350 to L, G, A, V, or I.
The disclosure further characterizes the modified endoinulinases as enzymes for inulin hydrolysis to fructooligosaccharides with tailored product profiles including DP3–DP6. Documented characterization includes improved enzyme properties versus recombinant wild type, including increased soluble expression and hydrolysis activity in cell lysates, and a shift in pH optimum to about 5.4, supported by biochemical characterization and product analysis.
Claims Coverage
The partial content includes one independent claim. The claim coverage is centered on a modified endoinulinase with a defined sequence identity requirement and specific residue substitutions at position 128, optionally combined with substitutions at positions corresponding to 316, 344, 350, or 504.
Modified endoinulinase with Y128 substitution and optional multi-site substitutions
A modified endoinulinase being a modified wild-type T. purpuregenus endoinulinase comprising an amino acid sequence set forth in SEQ ID NO: 1 with a substitution of tyrosine at position 128 (Y128) to H, K, or R or a functional fragment thereof, both comprising an amino acid sequence having at least 90% identity to a T. purpuregenus wild-type sequence of endoinulinase set forth in SEQ ID NO: 3, wherein any one of said modified endoinulinase or said functional fragment thereof optionally further comprises one or more amino acid substitutions in positions corresponding to position 316, 344, 350 or 504 of said wild-type T. purpuregenus endoinulinase.
Across the provided claims, protection is directed to modified endoinulinase sequences defined by Y128 substitution to H, K, or R and at least 90% identity to the wild-type sequence (SEQ ID NO: 3), optionally combined with additional substitutions at positions corresponding to 316, 344, 350, or 504.
Stated Advantages
Increased soluble expression compared to recombinant wild type.
Up to about 5-fold higher hydrolysis activity in cell lysates compared to recombinant wild type.
Shift in pH optimum to about 5.4.
Improved kinetic/catalytic metrics.
Mg2+-dependent activity increases.
Documented Applications
Producing fructooligosaccharides by contacting inulin with the modified endoinulinase or a functional fragment thereof.
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