Neutral endo-xylanase and encoding gene and application thereof
An endo-xylanase and neutral technology, applied in the fields of application, glycosylase, genetic engineering, etc. Systematic research on glycanase and other issues
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Embodiment 1
[0057] (1) Materials
[0058] 1. Strain: Streptomyces alkalophilus, Streptomycessp.H31. The strain preservation unit: China Typical Culture Collection Center (CCTCC), preservation time: January 5, 2015, preservation address: China. Wuhan. Wuhan University, preservation number: CCTCC NO: M2015003; this strain has been listed in the patent "201510034112.3 , a Streptomyces alkalophilus and the neutral endoglucanase produced by it and its application" are disclosed. E.coliTOP10F' was purchased from Invitrogen;
[0059] 2. Vector: the Escherichia coli cloning vector pMD18-T was purchased from Dalian Bao Biological Company, and the Escherichia coli expression vector pET-28a(+) (Novagen, KanR) was purchased from Novagen Company; see the vector map figure 1 , its multiple cloning site map is shown in figure 2 .
[0060] 3. Medium
[0061] (1) Selection medium: xylan 8.0g / L, KNO 3 1.0g / L, MgSO 4 ·7H 2 O0.5g / L, NaC115g / L, KH 2 PO 4 1.5g / L, solid medium plus agar 15-20g / L, pH...
Embodiment 2
[0128] (1) Materials
[0129] 1. Strains: Streptomyces alkalophilus Streptomycessp.H31 strain (CCTCC No: M2015003). The host bacteria E.coliBL21Star(DE3) and E.coliTOP10F' were purchased from Invitrogen;
[0130] 2. Vector: Escherichia coli expression vector pET-28a(+) (Novagen, Kan R ) was purchased from Novagen; vector map see Figure 9 , its multiple cloning site map is shown in Figure 10 .
[0131] 3. Medium and buffer
[0132] (1) Selection medium: xylan 8.0g / L, KNO 3 1.0g / L, MgSO 4 ·7H 2 O0.5g / L, NaC115g / L, KH 2 PO 4 1.5g / L, solid medium plus agar 15-20g / L, pH9.0;
[0133] (2) LB medium: tryptone 10g / L, yeast extract 5g / L, NaCl 10g / L, solid medium plus agar 15-20g / L, pH 9.0, autoclaved at 121°C for 20min.
[0134] (3) TE buffer: 10 mmol / LTris-Hcl, pH 8.0, 1 mmol / LEDTA, pH 8.0.
[0135] (4) Alkaline lysis solution I, II, III (plasmid extraction): Alkaline lysis solution I: glucose 50mmol / L, Tris-HCl (pH8.0) 25mmol / L, EDTA 10mmol / L. About 100mL per bottle, st...
Embodiment 3
[0209] The enzymatic property research of embodiment 3 neutral endoxylanases
[0210] (1) Experimental method
[0211] 1. Optimum reaction pH and pH stability
[0212] Take an appropriate amount of the pure enzyme solution of the neutral endoxylanase obtained in Example 2, add 1% xylan solutions with different pH values respectively, and measure the enzyme activity according to a conventional method. At the same time, the pure enzyme solution was stored at 37° C. for 60 min at different predetermined pH conditions, and then the remaining enzyme activity was measured at pH 7.0 according to a conventional method.
[0213] 2. Optimum reaction temperature and temperature stability
[0214] Take an appropriate amount of the pure enzyme solution of the neutral endoxylanase obtained in Example 2, place them in different predetermined temperature conditions and react for 20 minutes, and measure the enzyme activity. At the same time, the pure enzyme solution was placed under diffe...
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