Carboxyl magnetic bead immobilized glutamic acid decarboxylase and preparation method and application thereof

Abstract

The invention discloses carboxyl magnetic bead immobilized glutamic acid decarboxylase and a preparation method and application thereof. According to the carboxyl magnetic bead immobilized glutamic acid decarboxylase, carboxyl magnetic beads are adopted as a carrier, glutamic acid decarboxylase is fixed to the surface of the carboxyl magnetic beads through coupling of amidogen and carboxyl, and the base sequence of encoding genes of the glutamic acid decarboxylase is shown as SEQ ID NO.1. The preparation method comprises the following steps that 1, the glutamic acid decarboxylase and the carboxyl magnetic beads are provided; 2, the glutamic acid decarboxylase and the activated carboxyl magnetic beads are mixed, and after the reaction is completed, the carboxyl magnetic bead immobilized glutamic acid decarboxylase is obtained through separation. The carboxyl magnetic bead immobilized glutamic acid decarboxylase has the advantages of being easy to separate, capable of being repeatedly recycled, high in thermal stability, high in operation stability and the like. Compared with free enzymes, half-inactivation temperature of the carboxyl magnetic bead immobilized glutamic acid decarboxylase is improved by 2.1 DEG C, and after 10 times of repeated catalyzing, the activity is still kept at 90.42%.

Description

technical field

[0001] The invention relates to the technical field of molecular biology, in particular to a carboxyl magnetic bead immobilized glutamic acid decarboxylase and its preparation method and application. Background technique

[0002] Glutamate decarboxylase (GAD for short; EC4.1.1.15) is a class of pyridoxal phosphate-dependent enzymes that can specifically catalyze L-glutamine with pyridoxal phosphate (PLP) as a coenzyme Acid decarboxylation synthesizes γ-aminobutyric acid (γ-aminobutyrate, GABA), which is the key enzyme for biocatalytic preparation of GABA. GABA is a naturally occurring non-protein amino acid. It has many important physiological functions such as lowering blood pressure, calming the nerves, improving sleep and memory, and has broad application prospects in food, medicine, animal husbandry, agriculture and other fields. As a new type of functional factor, GABA is attracting more and more attention. It can be developed into a drug with significa...

Examples

Embodiment 1

[0046] 1. Preparation of engineered bacteria containing target gene

[0047] Utilize plasmid pET-28a(+) to construct recombinant plasmid pET-28a(+)-gad; use pET-28a(+) as the carrier, GAD1407 base sequence (as shown in SEQ ID NO.1) as the target fragment, after optimization Digestion, recovery, purification and ligation, construction of expression plasmids, transformation of the ligation reaction product into E.coli BL21(DE3), and the engineering bacteria E.coli BL21(DE3)-pET-28a(+) expressing glutamic acid decarboxylase GAD -GAD1407.

[0048] 2. Isolation and purification of glutamic acid decarboxylase

[0049] The preserved engineered bacteria were cultured in 5 ml of LB liquid medium containing 50 μg / mL kanamycin, and cultured overnight at 37° C. in a shaker at 200 rpm. Inoculate 2% of the inoculum into LB liquid medium containing 50 μg / mL kanamycin, shake and cultivate at 37°C and 200 rpm in a shaker, when the OD of the bacteria 600 When it reaches 0.6-0.8, add an appro...

technical field

[0001] The invention relates to the technical field of molecular biology, in particular to a carboxyl magnetic bead immobilized glutamic acid decarboxylase and its preparation method and application. Background technique

[0002] Glutamate decarboxylase (GAD for short; EC4.1.1.15) is a class of pyridoxal phosphate-dependent enzymes that can specifically catalyze L-glutamine with pyridoxal phosphate (PLP) as a coenzyme Acid decarboxylation synthesizes γ-aminobutyric acid (γ-aminobutyrate, GABA), which is the key enzyme for biocatalytic preparation of GABA. GABA is a naturally occurring non-protein amino acid. It has many important physiological functions such as lowering blood pressure, calming the nerves, improving sleep and memory, and has broad application prospects in food, medicine, animal husbandry, agriculture and other fields. As a new type of functional factor, GABA is attracting more and more attention. It can be developed into a drug with significa...

Examples

Embodiment 1

[0046] 1. Preparation of engineered bacteria containing target gene

[0047] Utilize plasmid pET-28a(+) to construct recombinant plasmid pET-28a(+)-gad; use pET-28a(+) as the carrier, GAD1407 base sequence (as shown in SEQ ID NO.1) as the target fragment, after optimization Digestion, recovery, purification and ligation, construction of expression plasmids, transformation of the ligation reaction product into E.coli BL21(DE3), and the engineering bacteria E.coli BL21(DE3)-pET-28a(+) expressing glutamic acid decarboxylase GAD -GAD1407.

[0048] 2. Isolation and purification of glutamic acid decarboxylase

[0049] The preserved engineered bacteria were cultured in 5 ml of LB liquid medium containing 50 μg / mL kanamycin, and cultured overnight at 37° C. in a shaker at 200 rpm. Inoculate 2% of the inoculum into LB liquid medium containing 50 μg / mL kanamycin, shake and cultivate at 37°C and 200 rpm in a shaker, when the OD of the bacteria 600 When it reaches 0.6-0.8, add an appro...

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