Study On Production, Clonging And Expression Of The Alginate Lyase From Bacillus Cereus

Alginates are linear (1-4)-linked polysaccharides which consist of the uranic acid,β-D-mannuronate and its C5 epimer, a-L-guluronate, and can be efficiently degraded by alginate lyase (ALGL) to produce alginate-oligosaccharide. Currently, there is an increasing demand for alginate-oligosaccharides obtained from alginates for its biotechnological and medical uses such as the anti-tumor effects which can promot the growth of bifidobacteria and the elongation of barley roots. In addition, world population outburst together with increased motorization has led to an overwhelming increase in the demand for fuel. Due to the limited supply of fossil petroleum and rapidly increased price, renewable bioenergy is becoming more attractive. Algae are a kind of carbohydrate feedstock, which might be served as an excellent alternative to food crops for bioethanol production, a green fuel for sustainable development. In order to turn algae into bioethanol, alginates must be degraded into alginate-oligosaccharides. However, the conversion of those alginates to fermentable alginate-oligosaccharides using alginate lyase is one of the most challenging issues.As an important enzyme for alginate-oligosaccharides production, the main source of ALGL is microorganism. Gene engineering techniques are commonly used to improve the enzyme producing microorganism to obtain large amount of alginate lyase. So far, approximately 20 genes encoding alginate lyases from bacteria have been cloned and sequenced. Among these 20 alginate lyases, most of them are substrate specified for only either poly-guluronate or poly-mannuronate. On the contrary, it was reported that the extracelluar alginate lyase produced by Bacillus sp. has substrate specificity for both poly-guluronate and poly-mannuronate units in the alginate molecule, which may be one of the promising enzymes for oligo-alginate production. However, no information was reported about cloning and expression of alginate lyase gene from Bacillus sp. In this paper, the cloning and expression of alginate lyase gene (algl) from Bacillus cereus in E.coli were described. And functional characterizations of the recombinant ALGL were presented.The study focused on the effects of eight principal elements on the enzyme producing abilities of strains, which concluding carbon concentration, nitrogen concentration, initial pH, temperature, volume of inoculum, content in flask, cultivation temperature and rotating speed through single-factor and response surface experiments to determine optimum conditions for enzyme production of strains. The compositions of fermentation medium are: the carbon concentration %, the nitrogen concentration 0.44%, pH 7.35. The optimal volume of inoculum is 4%, and cultivation temperature is 37°C, the optimal shaking speed is 160rpm when 250mL flask contains 65mL medium, culture time 42h. Under these conditions the enzyme activity can reach 6.7U/mL.The alginate lyase gene was amplified from the genomic DNA of Bacillus cereus F1-5-10 strain by using PCR with the primers obtained from the homolog sequences of alginate lyases. The predicted product of the algl insert revealed a single open reading frame (ORF) of 1032 bp encoding a polypeptide of 344 amino acid residues with a calculated molecular weight (MW) of 38.2 kDa including a predicted N-terminal signal peptide of 63 amino acid residues in length. The DNA sequence of the F1-5-10 alginate lyase was registered in the GenBank database with accession number GU585575. The amino acid sequence of the ALGL was aligned against other alginate lyases with the molecular weight less than 40 kDa (referred to as 40 Mr class) since the molecular weight of ALGL alginate lyase was 38.2 kDa, The ALGL alginate lyase was shown to have the none of the characteristic residues listed above based on multiple sequence alignment.Overexpression of the recombinant protein was performed by transforming the pET30a–algl to E.coli BL21 (DE3) strain. A distinct band of approximately 45 kDa, corresponding to the expected molecular weight of the His-tagged ALGL protein, was observed only after induction. According to the SDS-PAGE analysis, the His-tagged ALGL protein was partly soluble.Meanwhile, several expression parameters including induction times, IPTG concentrations and induction temperatures were tested to optimize the expression of the ALGL protein. The protein was found to have the highest expression with 0.4mM IPTG at 32°C for 4 h. ALGL exhibited a relatively high level of expression which constituted approximately 36% of the total proteins, analyzed by a densitometry scan of the 12%SDS-PAGE gel.The purification and biochemical characterization of the ALGL expressed in E.coli BL21 were investigated. The recombinant ALGL showed optimal the maximum activity at pH 6.6 and 35°C. Thus, the ALGL could be classified as a neutral and moderately thermostable alginate lyase. The Km and Vmax values were 1.89mg/ml and 15.01U/ml, respectively.