Glucamylase TlGA1931 and gene and application thereof
A glucoamylase and gene technology, applied in the field of agricultural biology, can solve the problem of low optimum temperature of glucoamylase, and achieve the effect of excellent production properties and good temperature stability
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Embodiment 1
[0049] Cloning of embodiment 1 glucoamylase coding gene Tlga1931
[0050] 1. Genomic DNA of Glucoamylase
[0051] PCR amplification was performed using the total DNA of the thermophilic cyanobacterium Talaromyces leycettanus JCM 12802 as a template, and the primers were shown in Table 1. The PCR reaction parameters were: 95°C for 5min; 94°C for 30sec, 50°C for 30sec, 72°C for 2min, 30 cycles , 72°C for 10 min. A fragment of about 1800bp was obtained, which was recovered and sequenced, and its nucleotide sequence is shown in SEQ ID NO.1.
[0052] Table 1 Primers required for gene cloning in this experiment
[0053]
[0054] 2. Acquisition of Glucoamylase cDNA
[0055] Extraction of total RNA from Talaromyces leycettanus JCM 12802, using Oligo(dT) 20 and reverse transcriptase to obtain a strand of cDNA, then utilize the primers 1931F and 1931R shown in Table 1 to amplify the single-stranded cDNA to obtain the cDNA sequence of glucoamylase, and obtain the sequenced after the...
Embodiment 2
[0057] The construction of embodiment 2 glucoamylase TlGA1931 engineering strain
[0058] 1. Construction and expression of expression vectors
[0059] Taking the cDNA fragment of the glucoamylase coding gene Tlga1931 that is sequenced correctly as a template, the coding region of its mature protein is amplified with primers 1931F and 1931R with EcoR I and Not I restriction sites shown in Table 1 . Use EcoR I and Not I to digest the PCR product, connect it into the expression vector pPIC9, insert the sequence of the mature protein of glucoamylase TlGA1931 into the downstream of the signal peptide sequence of the above expression vector, form a correct reading frame with the signal peptide, and construct a yeast expression The vector pPIC9-Tlga1931 was used to transform Escherichia coli competent cells Trans1. The positive transformants were subjected to DNA sequencing, and the transformants with the correct sequence were used for large-scale preparation of recombinant plasmi...
Embodiment 3
[0063] The preparation of embodiment 3 recombinant glucoamylase TlGA1931
[0064] 1. Mass expression of glucoamylase gene Tlga1931 in Pichia pastoris fermentation level
[0065] The transformants with higher enzyme activity were screened out, inoculated in YPD medium for activation and enrichment, and carried out mass expression at the fermentation level. The fermentation broth was centrifuged at 12,000×g for 10 min, the supernatant fermentation broth was collected, the enzyme activity was detected and analyzed by SDS-PAGE protein electrophoresis.
[0066] 2. Purification of Glucoamylase TlGA1931
[0067] The supernatant of the recombinant glucoamylase TlGA1931 expressed in the shake flask was collected, concentrated through a 10kDa membrane bag, and at the same time the medium was replaced with a low-salt buffer, and then further concentrated with a 10kDa ultrafiltration tube. Concentrate the recombinant glucoamylase TlGA1931 which can be diluted to certain times, and purif...
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