Cellulose Enzyme Directed Evolution, Site-directed Mutagenesis, And Alkali-resistant Mechanism

In recent years, important applications for cellulases in different industrial processes have been found. One major area of application is for the textile, detergent and the pulp and paper industries. All these industrial process are carried out at neutral or alkaline condition. So, alkaline or alkali-tolerant cellulases are preferred for these applications. A novel strain of Bacillus subtilis Y106 producing alkali-tolerant cellulase was isolated from alkaline soil. The gene for cellulase was cloned into plasmid pUC18. The recombinant plasmid was named pUC18-7 and expressed in E.coli JM109. The cellulase produced by E.coli JM109 (Cel 18-7), the same as by the original strain, exhibits the highest CMCase activity at pH6.0~6.5.Thus it is not very suitable for the demand of texile, laundry, and paper industries. In order to obtain alkaline or more alkali-tolerant cellulase, the directed evolution and site-directed mutagenesis were carried out in vitro to change the characteristics of cellulase.In this research, the error-prone PCR and mutator strain were used to establish the library of random mutant cellulase. By the technique of double-layered plate and the enzymatic assay of liquid culture, the mutant was screened. No mutant with the optimal pH of cellulase alkaline shift was screened from more than 600 mutants. But one mutant Cel 18-7-12 with optimal pH 6.0 was obtained, which had higher activity than that of the wild type in the acid condition. The results of sequencing showed that Ile in the 339 position was replaced by Thr, and there was a stop codon mutation caused 25 amino acids lost in C-terminal. In order to study the influence of CBD to the enzyme activity, 163 animo acids in the C-terminal were cut off using PCR method, and the recombinant enzyme was named CBDcut. The enzyme activeity of CBDcut increased in little extent, but it's pH profile was not changed. Therefore, the C-terminal.CBD region of cellulase might have little effect on the pH activity profile.The alignments of the amino acid sequence of 8 cellulases in family 5 were compared. The His-140 and Asn-212 of Cel 18-7 were found to be highly conserved. In this family, all acidic cellulase had a Gin-140 and Asn-212 while all basic enzyme had an Ile-140 and His-212 at this position. Highly conserved amino acid located at specific position in cellulase should play an important role in the structure and funcition of the enzyme, especially in the aspect of alkali-tolerance in this case.Sited-directed mutagenesis was carried out at H140 and N212 sites by recombinant PCR to study the role of these conserved residues. His-140 was replaced by Gin, and Asn-212 was replaced by His and Asp, respectively. The cellulase produced by E.coli JM109 harboing plasmid pUC18-7, pUC18-7-N212H, pUC18-7-N212D and pUC18-7-H140Q, respectively, was compared. The pH profiles of the corresponding mutant cellulases were quite different. The optimal pH of Cel 18-N212H had shifted from 6.5 to 7.0 and its activity in alkaline pH range increased, while Cel 18-N212D had shifted its optimal pH from 6.5 to 5.5 and lost its activity in alkaline pH range. The mutant Cel 18-H140Q showed higher activity than that of the wild type in the acid condition, but its activity in the alkaline range also increased.The figures of the 3D structure of the wild type and the mutated cellulases were constructed by Swiss-model, an automated homology modeling server running in the Geneva Glaxo Welcom Experimental Reseatch, Switzerland.The distance between the atoms of the side chain of the mutated amino acids and the carboxylic oxygen atoms of the catalytic residue were detected. The distance between the two catalytic residues in each cellulase was also detected. The alkali-tolerant mechanism of the cellulase was analyzed from the following aspects: the charged chatacter, the ionization state, and the hydrogen-bonding net, of the catalytic residues.The results of the present work should be very useful in the construction of high level expressing strains applied for the industry using site-directed mutagenesis methods, and also in the study of the relationship between the strcture and function of the cellulase,especially in the mechanism of the alkali-tolerance of cellulase.