US 12,410,412 B2
Method for producing trehalose employing a trehalose phosphorylase variant
Christopher David Bayer, Amsterdam (NL); Andreas Vogel, Leipzig (DE); Marc Struhalla, Leipzig (DE); and Birgit Brucher, Leipzig (DE)
Assigned to C-LECTA GMBH, Leipzig (DE); and NEW MATTERHORN, LLC, Wilmington, DE (US)
Filed by C-Lecta GmbH, Leipzig (DE); and New Matterhorn, LLC, Wilmington, DE (US)
Filed on Jul. 12, 2022, as Appl. No. 17/811,933.
Application 17/811,933 is a continuation of application No. 16/761,735, granted, now 11,421,207, previously published as PCT/EP2018/082881, filed on Nov. 28, 2018.
Claims priority of application No. 17204211 (EP), filed on Nov. 28, 2017; and application No. 18174349 (EP), filed on May 25, 2018.
Prior Publication US 2023/0193220 A1, Jun. 22, 2023
This patent is subject to a terminal disclaimer.
Int. Cl. C12N 9/10 (2006.01); C12N 9/92 (2006.01); C12P 19/12 (2006.01); C12P 19/18 (2006.01); C12P 19/24 (2006.01)
CPC C12N 9/1051 (2013.01) [C12N 9/92 (2013.01); C12P 19/12 (2013.01); C12P 19/18 (2013.01); C12P 19/24 (2013.01); C12Y 204/01007 (2013.01); C12Y 204/01064 (2013.01)] 21 Claims
OG exemplary drawing
 
1. A method for producing trehalose, comprising the steps of mixing and reacting, in any order,
a) at least one alpha-phosphorylase capable of catalyzing the production of alpha-D-glucose 1-phosphate intermediate from a saccharide raw material, where the alpha-phosphorylase is a sucrose phosphorylase and the saccharide raw material comprises sucrose, and from at least one phosphorus source selected from the group consisting of a phosphoric acid and an inorganic salt thereof;
b) at least one trehalose phosphorylase capable of catalyzing the production of trehalose from an alpha-D-glucose 1-phosphate intermediate and a glucose substrate, wherein the glucose substrate comprises glucose and the trehalose phosphorylase is a trehalose phosphorylase variant with an amino acid sequence which differs from the amino acid sequence of a wild type trehalose phosphorylase in at least one amino acid position;
c) at least one saccharide raw material which is sucrose and produces an alpha-D-glucose 1-phosphate intermediate and a fructose co-product by catalytic action of the alpha-phosphorylase;
d) at least one phosphorus source selected from the group consisting of a phosphoric acids and an inorganic salt thereof; and
e) optionally, at least one glucose isomerase,
wherein the trehalose phosphorylase variant comprises an amino acid sequence, wherein the amino acid sequence is at least 64% homologous and/or identical to the amino acid sequence of SEQ NO: 1, wherein the variant comprises amino acid substitutions at 2 to 15 of the amino acid positions selected from:
i. 712A, 712G, 712I, 712M, 712P or 712V, and/or
ii. 383A, 383G, 383I, 383L, 383M, 383V, 383N, 383C, 383Q, 383S or 383T, and/or
iii. 114A, 114G, 114I, 114M, 114P or 114V, and/or
iv. 118 is 118A, 118G, 118I, 118L, 118M, 118P or 118V, and/or
v. 225A, 225G, 225I, 225L, 225M, 225P or 225V, and/or
vi. 304G, 304I, 304L, 304M, 304P or 304V, and/or
vii. 323A, 323G, 323I, 323L, 323M, 323P, or 323V, and/or
viii. 349W or 349Y, and/or
ix. 357A, 357I, 357L, 357M, 357P or 357V, and/or
x. 487A, 487G, 487I, 487L, 487M, 487P or 487V, and/or
xi. 550A, 550G, 550I, 550L, 550M or 550P, and/or
xii. 556N, 556C, 556Q or S556T, and/or
xiii. 564D or 564E, and/or
xiv. 590N, 590C, 590Q, 590S, 590T, 590A, 590G, 590I, 590L, 590M, 590P or 590V, and/or
x. 649D or 649E,
wherein the amino acid numbering refers to an aligning position in SEQ ID NO: 1.