Alkaline Cellulase Producing Strain, Cloning And Expression In Escherichia Coli

The study on cellulose resources and cellulases has become a current research hotspot and received intensive attention all over the world. Among the various types of cellulases widespread in nature, the bacterial alkaline cellulases are of great importance and extensively applied in detergent, textile, paper manufacturing, environmental protection, and many other industrial fields.More than 20 bacteria strains producing alkaline cellulases were isolated from alkaline soil samples, among which, the alkalophilic bacterium Z-16, exhibited highest cellulase activity. Phylogenetic analysis based on 16S rDNA gene sequence together with part of physiological and biochemistry characteristics indicated that Z-16 belonged to the genus of Bacillus. The highest CMCase activity of the crude cellulase produced by Bacillus sp. Z-16 was obtained at 50℃and pH 7.0-7.5, and approx.80% of the activity was within pH 6.5-9.0. This enzyme showed excellent pH stability and thermostability. It retained more than 90% of its maximal activity for 2 h at pH 3-11. The activity of this enzyme was activated by Na+, K+ and Mn2+, but inhibited by Co2+, Hg2+, Cd2+ and SDS. Whereas Zn2+ and Cu2+ caused deactivation of this enzyme, but EDTA, Triton X-100 and Tween 20 had no significant effect on the activity.The growth conditions that influenced the cellulase production of Bacillus sp. Z-16 were investigated and optimized. When CMC-Na was used as carbon source and yeast extract as nitrogen source, initial pH 9.0,10% (v/v) medium volume,3% (v/v) inoculum,30℃and shaken at 120 rpm for 36 h, the highest cellulase activity reached 3.48 U/mL, which was 7.5 times higher than its initial value.Primers were designed based on the published sequences of cellulase genes from homologous strains. The cellulase gene fragment of Bacillus sp. Z-16 was amplified by using its chromosomal DNA as template and sequenced. Its ORF consists of 2475 nucleotides and encodes a protein composed of 824 amino acids. The calculated molecular weight was 88,181 daltons and predicted pI was 4.70. According to the analysis of its amino acids, this enzyme consists of a signal peptide (30 amino acids), a glycosyl hydrolase family five domain at its N terminus, and a CBM₁7₂8 cellulose binding domain at its C terminus. The catalytic domain of the cellulase shows 72% identity with Cel K, an alkaline cellulase applied in detergent industry in Japan.The gene (gen 2) encoding for cellulase without signal peptide and the gene (gen 3) encoding for the catalytic domain were cloned and inserted into pET-28a(+), then expressed in E. coli BL21 (DE3), respectively. The condition for expressing the corresponding recombinant cellulase, named Cel 2 and Cel 3, was optimized, respectively.0.8 mM of IPTG was added when the cell density reached 0.8 of OD600 followed by 16 hours of cultivation at 37℃, the extracellular activities of Cel 2 and Cel 3 reached 1.191 U/mL and 9.307 U/mL, and the specific activities were 17.80 U/mg and 45.13 U/mg, respectively.The extracellular Cel 2 and Cel 3 were concentrated by ultrafiltration. After the procedure of affinity chromatography, they were purified 3.2-fold and 2.5-fold with specific activities of 63.5 U/mg and 89.0 U/mg, respectively. The maximum CMCase activity of Cel 2 was measured at 50℃and pH 7.5, and 90% of the activity was obtained at pH 7.5-9.0, which was similar with wild-type enzyme. However, the maximum CMCase activity of Cel 3 was measured at 55-60℃and pH 7.5, and 90% of the activity at pH 6.5-8.0, which exhibited a acidic shift of 1.0 pH unit compared to Cel 2 and the wild-type enzyme. The effects of metal ions, EDTA and surfactants on the activity of Cel 2 and Cel 3 were the same as that on the wild-type enzyme. The Km and Vmax of purified Cel 2 and Cel 3 were also measured. The Km value of Cel 2 was 0.105 mmol/L, and 0.900 mmol/L for Cel 3. The Vmax of Cel 2 was 0.05 mmol/L/min and 0.280 mmol/L/min for Cel 3.In this reaserch, an alkalophilic bacillus producing alkaline cellulase with excellent pH stability and thermostability was isolated. The gene encoding for the cellulase was cloned and expressed in E.coli. The recombinant cellulase and its CD domain were purified, and studied. This work not only provides a good material for the study of alkali-tolerrance mechanism of cellulase, but also benefits for the application of alkaline cellulase.