A membranous immobilized cell, polypeptide, oligopeptide or protein and a preparation method thereof are provided. The method includes the following steps: 1) providing un-film-form chitosan, where the chitosan is un-pre-crosslinked or pre-crosslinked; 2) providing a mixture of the un-film-form chitosan and cell, polypeptide, oligopeptide or protein, and in the mixture, the un-film-form chitosan is in a dissolved state; 3) mixing the mixture with a crosslinking reagent to obtain a co-crosslinked product of the chitosan and the cell, polypeptide, oligopeptide or protein; and 4) drying the co-crosslinked product to obtain membranous immobilized cell, polypeptide, oligopeptide or protein. When un-pre-crosslinked chitosan is used in the step 1), the method further includes comprises the step 5) mixing the membranous immobilized cell, polypeptide, oligopeptide or protein with phosphate salt, so that chitosan molecules therein are crosslinked with each other.

An object of the present invention is to provide a rare sugar-containing composition having improved properties as a sweetener while maintaining the excellent functionality of D-psicose. The invention relates to a method of producing a rare-sugar containing composition with D-fructose as a raw material. An intended product having a novel composition of D-glucose, D-fructose, and D-psicose is obtained by preparing a mixture of D-fructose and D-psicose by using ketose 3-epimerase in the first stage and converting D-fructose into D-glucose by using D-xylose isomerase inert to D-psicose in the second stage. The first-stage product is a mixture of D-fructose and rare sugar D-psicose. The resulting mixture is a sweetener having a taste quality equal to that of sugar.

The present invention relates to polypeptide, preferably to a glucose isomerase, comprising an amino acid sequence, wherein the amino acid sequence of the polypeptide, preferably the glucose isomerase, is at least 95% identical to an amino acid sequence of SEQ ID NO:1, wherein the amino acid sequence of the polypeptide, preferably the glucose isomerase, comprises an amino acid substitution at one or more amino acid positions, wherein the one or more amino acid positions is/are each and independently selected from the group consisting of SEQ ID NO: 1 amino acid positions 89, 90, 95, 0, 33, 34, 35, and 59. The present invention further relates to methods preparing glucose including the use of the polypeptides of the invention for preparing glucose.

The present invention relates to polypeptide, preferably to a glucose isomerase, comprising an amino acid sequence, wherein the amino acid sequence of the polypeptide, preferably the glucose isomerase, is at least 95% identical to an amino acid sequence of SEQ ID NO:1, wherein the amino acid sequence of the polypeptide, preferably the glucose isomerase, comprises an amino acid substitution at one or more amino acid positions, wherein the one or more amino acid positions is/are each and independently selected from the group consisting of SEQ ID NO: 1 amino acid positions 89, 90, 95, 0, 33, 34, 35, and 59. The present invention further relates to methods preparing glucose including the use of the polypeptides of the invention for preparing glucose.

The present invention relates to a yeast cell that is genetically modified comprising: a) a disruption of one or more aldehyde dehydrogenase (E.C: 1.2.1.4) native to the yeast; b) one or more nucleotide sequence encoding a heterologous NAD+-dependent acetylating acetaldehyde dehydrogenase (E.C. 1.2.1.10); c) one or more nucleotide sequence encoding a homologous or heterologous acetyl-CoA synthetase (E.C. 6.2.1.1); and d) a modification that leads to reduction of glycerol 3-phosphate phosphohydrolase (E.C. 3.1.3.21) and/or glycerol 3-phosphate dehydrogenase (E.C. 1.1.1.8 or E.C. 1.1.5.3) activity, native to the yeast.

The present invention relates to a yeast cell that is genetically modified comprising: a) a disruption of one or more aldehyde dehydrogenase (E.C: 1.2.1.4) native to the yeast; b) one or more nucleotide sequence encoding a heterologous NAD+-dependent acetylating acetaldehyde dehydrogenase (E.C. 1.2.1.10); c) one or more nucleotide sequence encoding a homologous or heterologous acetyl-CoA synthetase (E.C. 6.2.1.1); and d) a modification that leads to reduction of glycerol 3-phosphate phosphohydrolase (E.C. 3.1.3.21) and/or glycerol 3-phosphate dehydrogenase (E.C. 1.1.1.8 or E.C. 1.1.5.3) activity, native to the yeast.

The present invention relates to polypeptide, preferably to a glucose isomerase, comprising an amino acid sequence, wherein the amino acid sequence of the polypeptide, preferably the glucose isomerase, is at least 95% identical to an amino acid sequence of SEQ ID NO:1, wherein the amino acid sequence of the polypeptide, preferably the glucose isomerase, comprises an amino acid substitution at one or more amino acid positions, wherein the one or more amino acid positions is/are each and independently selected from the group consisting of SEQ ID NO: 1 amino acid positions 89, 90, 95, 0, 33, 34, 35, and 59. The present invention further relates to methods preparing glucose including the use of the polypeptides of the invention for preparing glucose.

The present invention relates to polypeptide, preferably to a glucose isomerase, comprising an amino acid sequence, wherein the amino acid sequence of the polypeptide, preferably the glucose isomerase, is at least 95% identical to an amino acid sequence of SEQ ID NO:1, wherein the amino acid sequence of the polypeptide, preferably the glucose isomerase, comprises an amino acid substitution at one or more amino acid positions, wherein the one or more amino acid positions is/are each and independently selected from the group consisting of SEQ ID NO: 1 amino acid positions 89, 90, 95, 0, 33, 34, 35, and 59. The present invention further relates to methods preparing glucose including the use of the polypeptides of the invention for preparing glucose.

Recombinant strains are obtained for the production of allulose, allose, and allitol by regulating intracellular glucose metabolism, reducing the enzyme activity of fructose 6-phosphate kinase, and enhancing the enzyme activities of glucokinase and glucose-6-phosphate isomerase, allulose 6-phosphate 3-epimerase, allulose 6-phosphate phosphatase, fructose permease and fructokinase, and optionally enhancing the enzyme activities of ribose 5-phosphate isomerase, allose 6-phosphate phosphatase, ribitol dehydrogenase, glycerol permease, glycerol dehydrogenase, and dihydroxyacetone kinase. A method for producing allulose and allose is an extracellular multienzyme cascade method. Multienzyme cascade catalysis and fermentation are coupled to improve the conversion rate of starch sugar or sucrose to the synthesized allulose.

Recombinant strains are obtained for the production of allulose, allose, and allitol by regulating intracellular glucose metabolism, reducing the enzyme activity of fructose 6-phosphate kinase, and enhancing the enzyme activities of glucokinase and glucose-6-phosphate isomerase, allulose 6-phosphate 3-epimerase, allulose 6-phosphate phosphatase, fructose permease and fructokinase, and optionally enhancing the enzyme activities of ribose 5-phosphate isomerase, allose 6-phosphate phosphatase, ribitol dehydrogenase, glycerol permease, glycerol dehydrogenase, and dihydroxyacetone kinase. A method for producing allulose and allose is an extracellular multienzyme cascade method. Multienzyme cascade catalysis and fermentation are coupled to improve the conversion rate of starch sugar or sucrose to the synthesized allulose.