Kluyveromyces lactis for producing neutral lactase
A technology of kluyveromyces and lactase, applied in the fields of bioengineering technology and enzyme engineering, can solve problems such as high cost and complicated extraction process, and achieve high safety effects
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Embodiment 1
[0047] Example 1: Screening of wild-type Kluyveromyces lactis strains
[0048] (1) Construction of a high-throughput screening method for lactase-producing strains:
[0049] The lactase substrate 4-methylumbelliferyl-β-D-galactopyranoside (MUGal) was selected according to the economic cost of the fluorescent substrate and the fluorescence sensitivity after the reaction. The applicability of K.lactis screening for high enzyme production: (1) The fluorescent substrate 4-methylumbelliferone-β-D-galactoside (MUGal) can be transported into K.lactis cells and degraded by lactase to generate fluorescent 4-methylumbelliferone (4-MU); (2) Lactase reacted with the fluorogenic substrate MUGal for a period of time at 30°C, in the range of 0~23U / mL, the fluorescence and the enzyme activity had a linear relationship; 0~23U / mL; Within the range of 50U / mL, fluorescence and enzyme activity were correlated; (3) K.lactis was inoculated into lactose medium containing MUGal, and intracellular flu...
Embodiment 2
[0057] Example 2: Identification of Lactase and Characterization of Enzymatic Properties
[0058] 1.1 The amino acid sequence of the enzyme
[0059] The genome of Kluyveromyces lactis JNXR-2101 screened in Example 1 was extracted, sequenced and analyzed. The results showed that the neutral lactase produced by Kluyveromyces lactis JNXR-2101 had the amino acid sequence shown in SEQ ID NO.1.
[0060] 1.2 Determination of specific enzyme activity of enzymes
[0061] The crude enzyme solution of Kluyveromyces lactis JNXR-2101 prepared according to the method of Example 1 was diluted with phosphate buffer solution (pH 6.5) to a certain gradient, and the enzyme activity was determined to be 28.89 U / mL after the reaction at 30 ° C for 10 min, and The protein concentration of the crude enzyme solution was determined to be 8.33 mg / mL using the BCA protein concentration detection kit. After conversion, the lactase specific enzyme activity of JNXR-2101 was 3.47 U / mg.
[0062] 1.3 The op...
Embodiment 3
[0070] Example 3: Kluyveromyces lactis JNXR-2101 utilizes different carbon sources to produce enzymes
[0071] Carbon source provides the energy required for microbial fermentation and the carbon skeleton for cell division, and is one of the basic nutrients for cell growth. The type and concentration of carbon source have a great impact on the production of extracellular enzymes. Glucose, lactose, galactose, sucrose, fructose, maltose, glycerol and soluble starch were selected as the only carbon source, the carbon source concentration was 20g / L, and the other components were the same as the YPL medium (i.e. tryptone 20g / L). , yeast powder 10g / L), natural pH value, prepare the seed liquid according to the method of Example 1, inoculate the seed liquid with 2% inoculum, ferment for 60h under the conditions of 30 ℃, 220rpm, and detect the intracellular and extracellular lactase activity of the fermented liquid . The determined optimal carbon source was added to the medium at fin...
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