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Lactase enzymes with improved activity at low temperatures

A technology of galactosidase and lactose, applied in the field of reducing the lactose content of dairy products, can solve the problems of low microbial count and unsuitability

Pending Publication Date: 2020-01-10
CHR HANSEN AS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Moreover, these lactases are not suitable for the hydrolysis of lactose in milk carried out at high or low temperatures, which in some cases is beneficial to keep microbial counts low and thus ensure high milk quality
Furthermore, known lactases are not suitable for use in the desired process of producing ultra heat treated (UHT) milk, where the enzyme is added prior to UHT treatment

Method used

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  • Lactase enzymes with improved activity at low temperatures
  • Lactase enzymes with improved activity at low temperatures
  • Lactase enzymes with improved activity at low temperatures

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0117] Example 1: Construction of an expression vector for producing lactase

[0118] Genomic DNA of lactic acid bacteria or bifidobacteria was extracted using a commercial genome extraction kit following the provided protocol (DNeasy, Qaigen, Germany). Using two synthetic primers, the lactase gene was amplified by PCR with a purified genomic DNA source as biomass, and the PCR reagent was provided with the Phusion U Hot start DNA polymerase (Thermo Scientific, USA) kit. The lactase gene was cloned into the start codon of the expression vector pBAD / His using the USER cloning method (Nour-Eldin HH, Geu-Flores F, Halkier BA, Plant Secondary Metabolism Engineering, Methods in Molecular Biology, 643; 2010), generating Expression constructs. Using the USER cloning method, long complementary overhangs are generated on both the PCR product and the destination vector. These overhangs can anneal to each other to form stable hybrid products for transformation into E. coli without lig...

Embodiment 2

[0119] Example 2: Expression of lactase in Escherichia coli expression host

[0120] Lactase was produced in E. coli BW25113 using the pBAD expression system. Collect freshly transformed E. coli BW25113 cells carrying the plasmid DNA from the Lb-Amp plate using a sterile loop and use to inoculate 5 mL of Lb-Amp medium. Overnight grown cultures (200 μL) were grown on a shaker ( Inoculate 50 mL of 2x PY medium (containing 100 μg / mL ampicillin) in a 250 mL flask in 42). Cultures were grown at 37°C, 220 rpm until an OD600 of 0.6-0.8 was reached. Lactase expression was initiated by adding 0.05% arabinose to the medium, and the cells were incubated for an additional 16-20 hours at 18°C, 180 rpm. Cells were harvested by centrifugation (5000 rpm, 10 minutes at 4°C) and stored at -20°C until further use.

Embodiment 3

[0121] Example 3: Protein Purification Using Immobilized Metal Affinity Chromatography

[0122] Thaw cells from a 50 mL culture on ice and use 10 mL of a mixture of lysis buffer ( (Novagen), containing 2 mg / mL lysozyme (Sigma Aldrich), 1 unit totipotent nuclease (Benzonase) (Sigma Aldrich), and 1X complete protease inhibitor cocktail (Complete Protease inhibitor cocktail) (without EDTA, Roche)), by adding Cells were lysed by incubating at room temperature for 30 minutes. After 30 minutes, cell debris was removed by centrifugation at 16000 rpm for 20 minutes at 4°C. The resulting supernatant was filtered through a filter with a pore size of 0.45 μm. A gravity flow Ni-Sepharose (GE Healthcare) column was prepared with 1 mL of the slurry by washing out ethanol and water. Then wash buffer (50mM NaH 2 PO 4 , pH 8.0, containing 300 mM NaCl and 20 mM imidazole) equilibrated the column. The cell-free extract is loaded onto the column and unbound protein is eluted from the col...

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Abstract

The present invention relates to new improved peptide or dimeric peptides exhibiting beta-galactosidase enzyme activity as well as improved methods for reducing the lactose content in compositions inparticular at low temperatures.

Description

technical field [0001] The present invention relates to a method of producing dairy products and a method of reducing the lactose content of dairy products using novel peptides or dimeric peptides exhibiting β-galactosidase activity and having improved activity at low temperatures. Background technique [0002] For growth in milk, lactose hydrolysis is a good way for lactic acid bacteria to obtain glucose and galactose as carbon sources. Lactase (β-galactosidase; EC 3.2.1.23) is an enzyme that hydrolyzes the sugar lactose in milk into monosaccharides. Lactase is used commercially to break down lactose in dairy products. People who are lactose intolerant have difficulty digesting dairy products with high lactose levels. It is estimated that around 70% of the world's population has a limited ability to digest lactose. Consequently, the demand for dairy foods that contain no or only low levels of lactose is growing. [0003] Lactase has been isolated from a variety of organ...

Claims

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Application Information

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IPC IPC(8): C07K14/195C12N9/38
CPCC07K14/195C12Y302/01023C12N9/2471A23C9/1206
Inventor H·拉杰P·史密斯T·埃克哈特V·沃因诺维奇C·E·G·苏勒约翰尼斯·马腾·范丹尼布林克
Owner CHR HANSEN AS
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