Gene Cloning, Expression, And Characterization Of Xylanases From Thermophilic Bacteriums

Xylanase plays an important role in the xylanolytic enzyme system by randomly cleaving theβ-1,4-glycoside linkages in xylan backbone and generating xylose and oligoxylose units with various branched substitutions. Many studies have been conducted to explore xylanases from various sources due to their widespread applications in the paper, food, textile and animal feed industries. Xylanases from the thermophilic microorganisms have favorable properties, thus the researches on these xylanases have been attracting much attention all over the world. The objective of this study is to isolate some thermophilic microorganisms, obtain xylan-degrading enzymes from them, and achieve high-yield expression of the recombinant enzymes.In this study, three thermophilic strains were isolated from the outflow of a hot spring in Baoshan city, Yunnan province, China. These bacteria were named as A4, A15 and E2, respectively. Based on sequence comparison of the 16S ribosomal deoxyribonucleic acid (rDNA), A4 and A15 were classified into the genus Alicyclobacillus, and designated as Alicyclobacillus sp. A4 and Alicyclobacillus sp. A15, respectively. Strain E2 belonged to the genus Anoxybacillus and designated as Anoxybacillus sp. E2. Strain A4 and A15 showed the optimal growth at 60°C and pH 3.0, and the optimal growth of strain E2 occurred at 60°C and pH 7.0–10.0 (still survival at pH 12.0).Three xylanase genes were cloned from the strains described above and expressed in Escherichia coli BL (DE3), respectively. XynA4-2 from Alicyclobacillus sp. A4 exhibited maximum activity at pH 6.2 and 55°C. The enzyme was thermostable at 50°C. When combined with the commercial glucanase from Sunson, XynA4-2 could increase the filtration rate and reduce the viscosity of mash. These favorable properties make XynA4-2 a good candidate in the brewing industry.XynA15 from Alicyclobacillus sp. A15 showed the maximum activity at 50°C and pH 7.0, was highly active over a broad temperature range (35–60°C) and highly thermostable at 50°C. Thus XynA15 had potential for applications in the paper, biofuel, and animal feed industries. XynE2 from Anoxybacillus sp. E2 showed maximal activity at pH 7.8 and 65°C, and was thermostable at 60°C. The enzyme was highly active and stable over a broad pH range, showing more than 90% of maximal activity at pH 6.6–8.6 and retaining more than 80% of activity at pH 4.6–12.0, 37°C for 1 h, respectively. These favorable properties make XynE2 a good candidate in the pulp and paper industries.In summary, three thermophilic bacterial strains were isolated from a high-temperature environment, and xylanase genes were cloned from the genomic DNA of each strain. These genes were heterologously expressed and characterized. All recombinant enzymes showed favorable properties for potential applications.