Heat-resistant beta-mannanase and its coding gene, recombinant bacterium and use
A technology of recombinant bacteria and genes, applied in the fields of application, genetic engineering, plant genetic improvement, etc., can solve the problems of being unable to resist feed pelleting temperature, poor heat resistance, and limiting the use of β-mannanase, and achieve a catalytic pH value Wide range, good acid stability and high specific activity
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Embodiment 1
[0037] Example 1, Discovery of thermostable β-mannanase (MANN-cx1 protein) and its coding gene
[0038] 1. Error-prone PCR amplification of β-mannanase gene
[0039] Using the optimized wild-type mannanase gene as a template, the error-prone PCR reaction system contains: dNTP (2.5mM), dCTP (10mM), dTTP (10mM), Mg 2+ (25mM), Mn 2+ (10 mM), Taq DNA polymerase (5 U). Primers were E: 5′-CCG GAA TTC TTG CCA AAG GC-3' (the underline is the restriction endonuclease EcoR I site), X: 5'-GC T CTA GA T TAA GCA GAA TC-3' (the underline is the restriction endonuclease XbaI cutting site). The PCR reaction conditions were: denaturation at 94°C for 1 min, annealing at 52°C for 30 s, extension at 72°C for 1 min, and 30 cycles. PCR products were detected by 1% agarose gel electrophoresis.
[0040] 2. Construction and screening of mutant expression library
[0041] The error-prone PCR product was digested with restriction endonucleases EcoR I and Xba I, and then connected to the self-r...
Embodiment 2
[0046] Embodiment 2, the construction of recombinant bacteria and the expression of thermostable β-mannanase (MANN-cx1 protein)
[0047] 1. Construction of engineering bacteria
[0048] 1. Synthesize the DNA (MANN-cx1 gene) shown in sequence 2 of the sequence listing.
[0049] 2. Using the MANN-cx1 gene synthesized in step 1 as a template, perform PCR amplification with a primer pair composed of F1 and R1 to obtain a PCR amplification product.
[0050] F1: 5'-CCG GAA TTC TTG CCA AAG GC-3' (underlined EcoR I restriction recognition sequence);
[0051] R1: 5'-GC T CTA GA T TAA GCA GAA TC-3' (Xba I restriction recognition sequence is underlined).
[0052] 3. The PCR amplified product of step 2 was double-digested with restriction enzymes EcoR I and Xba I, and the digested product was recovered.
[0053] 4. Digest the vector pPICZαA with restriction endonucleases EcoR I and Xba I to recover the vector backbone (about 3600 bp).
[0054] 5. Ligate the digested product of st...
Embodiment 3
[0083] Example 3, Enzymatic property analysis of thermostable β-mannanase (MANN-cx1 protein)
[0084] 1. Determination of the optimum pH value
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