Search

CN-121249640-B - Cellobiose epimerase mutant for high yield of lactulose and application thereof

CN121249640BCN 121249640 BCN121249640 BCN 121249640BCN-121249640-B

Abstract

The invention discloses a cellobiose epimerase mutant for high yield of lactulose and application thereof, and belongs to the technical field of enzyme engineering. The invention discloses a cellobiose epimerase (Disp-WT enzyme for short) from microorganism Dictyoglomus sp as parent, by using a gene mutation technology, the 245 th histidine His is replaced by glutamic acid Glu to obtain a mutant H245E. The optimal catalysis condition of the mutant enzyme H245E is not changed, but the isomerase activity of the enzyme at 85 ℃ is improved to 254% of the original activity, and the generation amount of the epalactose is greatly reduced. This finding is of great research value for the study of more cellobiose epimerases with lower isomerase activity.

Inventors

  • WEI ZHE
  • LV HOUCHEN
  • MU WANMENG
  • NI DAWEI
  • CHEN XINXIN
  • LIU QIANQIAN

Assignees

  • 山东金洋药业有限公司

Dates

Publication Date
20260508
Application Date
20251205

Claims (10)

  1. 1. A cellobiose epimerase mutant for producing lactulose is characterized in that the mutant is obtained by mutating histidine at 245 th position of cellobiose epimerase with an amino acid sequence shown in SEQ ID NO. 2 into glutamic acid.
  2. 2. A gene encoding the cellobiose epimerase mutant of claim 1.
  3. 3. A recombinant vector carrying the gene of claim 2.
  4. 4. A recombinant cell expressing the cellobiose epimerase mutant of claim 1, carrying the gene of claim 2, or carrying the recombinant vector of claim 3, wherein the recombinant cell is an expression host of bacteria or fungi.
  5. 5. A recombinase catalyst comprising the cellobiose epimerase mutant of claim 1, characterized by being in any one of the following forms: (1) Culturing a recombinant expression transformant containing the cellobiose epimerase mutant, and isolating a transformant cell containing the recombinant cellobiose epimerase mutant enzyme; (2) Culturing a recombinant expression transformant containing the cellobiose epimerase mutant, separating transformant cells containing the recombinant cellobiose epimerase mutant enzyme, and disrupting the transformant cells containing the recombinant cellobiose epimerase mutant enzyme to obtain a cell disruption solution; (3) Culturing recombinant expression transformant containing the cellobiose epimerase mutant, separating transformant cells containing recombinant cellobiose epimerase mutant enzyme, disrupting transformant cells containing recombinant cellobiose epimerase mutant enzyme to obtain cell disruption solution, and freeze-drying the cell disruption solution of recombinant cellobiose epimerase mutant enzyme to obtain freeze-dried enzyme powder.
  6. 6. A method for improving the enzyme activity of cellobiose epimerase or improving the yield of lactulose prepared by using cellobiose epimerase and reducing the byproduct epalactose is characterized in that histidine at 245 th position of cellobiose epimerase with an amino acid sequence shown as SEQ ID NO.2 is mutated into glutamic acid.
  7. 7. A method for increasing lactulose yield and reducing by-product epalactose, characterized in that the method comprises adding the cellobiose epimerase mutant according to claim 1, the recombinant cell according to claim 4, or the recombinase catalyst according to claim 5 to a reaction system containing lactose for reaction.
  8. 8. A method for preparing lactulose, characterized in that the cellobiose epimerase mutant according to claim 1, the recombinant cell according to claim 4, or the recombinase catalyst according to claim 5 is added into a reaction system containing lactose for reaction.
  9. 9. The method according to claim 8, wherein the cellobiose epimerase mutant is added in an amount of 0.05-2 mg/mL, the reaction condition is that the pH is 7.0-8.0, the temperature is 75-85 ℃, the reaction time is 15-120 min, and the lactose concentration is 100-800 mM.
  10. 10. Use of the mutant according to claim 1, or the gene according to claim 2, or the recombinant vector according to claim 3, or the recombinant cell according to claim 4, or the recombinase catalyst according to claim 5, for the preparation of lactulose or lactulose-containing products.

Description

Cellobiose epimerase mutant for high yield of lactulose and application thereof Technical Field The invention relates to a cellobiose epimerase mutant for high yield of lactulose and application thereof, belonging to the technical field of enzyme engineering. Background Lactose (Lactose; 4-O-beta-D-galactopyranosyl-D-glucose; C 12H22O11) is a disaccharide consisting of galactose and glucose, a carbohydrate commonly found in mammalian milk. Both monosaccharides constituting it are six-membered ring aldoses, lactose is hydrolyzed in water, and the glucose moiety is hydrolyzed to a ring-opened form, which is reducing. Only a very small fraction (< 0.1%) of lactose in the solution is in the open-loop state, but this fraction is extremely important, and they are in the chemically active state and are subject to chemical changes such as maillard reactions. Lactose has a low solubility compared to other disaccharides. At 25 ℃, its solubility is only one tenth of sucrose. However, lactose does not readily crystallize despite its low solubility, and only when supersaturation exceeds 2.1 will crystals precipitate. Crystalline lactose exists in a closed-loop form in two anomeric forms, alpha-lactose and beta-lactose, respectively. The alpha-lactose after crystallization exists in the form of a monohydrate, which is the main existing form of lactose in dry foods. Beta-lactose is crystallized in anhydrous form. Lactulose (4-O-beta-D-galactopyranosyl-D-fructose; C 12H22O11) is formed by connecting galactose and fructose through beta-1, 4 glycosidic bond. The white powder solid at normal temperature has good stability, high safety, easy dissolution in water and sweetness lower than lactose. Lactulose is not easily digested by the small intestine, can increase peristalsis of the human small intestine, can adsorb moisture and promote food to pass through the intestinal tract, and is applied to laxative drugs for half a century ago. In human colon, lactulose can provide energy for lactobacillus and bifidobacterium, and organic acids such as lactic acid and formic acid can be produced in the metabolic process, so that the balance of flora in intestinal tracts can be regulated, and mineral absorption is promoted. In addition, lactulose has the beneficial effects of treating hepatic encephalopathy, reducing blood ammonia concentration, promoting B vitamins generation, resisting endotoxin and the like. Just because of these properties of lactulose, it can also be used in foods as a functional food additive. For example, a formula containing 0.5% lactulose can stimulate the growth of bifidobacteria in the intestinal tract of infants, and a formula containing 1% lactulose has a laxative effect, and the addition of lactulose does not affect the storage quality of the formula. The yogurt containing the lactulose can also relieve constipation of infants, and the effect of the lactulose is better than that of soybean fibers and lactose oligosaccharides. The lactulose can shorten the fermentation of the yoghurt because the proliferation of the bifidobacterium microorganisms can be promoted. The industrial preparation method of lactulose at the present stage is mostly based on loberide-brucine-Fan (Lobry de Bruyn-VAN EKENSTEIN reaction), and common catalysts include hydroxide, tertiary amine compounds, boric acid, potassium carbonate and the like, lactose is used as a raw material in the reaction, lactose, lactulose, epinastine and the like are contained in the generated product, and the subsequent separation process is complex. In the food industry, enzymatic processes provide a great advantage over traditional chemical processes for preparing functional sugars. The enzymatic preparation has the characteristics of high conversion rate, strong specificity, simple operation procedure and the like, has more outstanding performance than the chemical preparation in the aspects of safety and environmental problems, and is also the trend of preparing functional sugar at present. Cellobiose epimerase is the only biocatalyst currently available for the efficient preparation of epalactose, and is the most specific biocatalyst to date for the preparation of lactulose. The cellobiose epimerase does not require the addition of the co-substrate fructose to lactose, and has higher conversion efficiency and fewer byproducts than other lactulose producing enzymes. Although there are already enzymes that can produce lactulose, cellobiose epimerase has a low specific enzyme activity, and since the yield of by-product epalactose is high, the cost of post-separation and purification is increased, so that it is necessary to perform molecular modification to increase the enzyme activity and reduce the yield of by-product epalactose. Disclosure of Invention The invention provides a cellobiose epimerase mutant, which is obtained by mutating 245 th amino acid of cellobiose epimerase with an amino acid sequence shown as SEQ ID NO. 2. In one embodiment of the inv