Expression, Processing And Export Of Pseudoalteromonas Elyakovii Alginate Lyase In Escherichia Coli, Purification And Characterization Of The Recombinant Alginate Lyase

In recent years, marine drugs have been developing very rapidly with the development and utilization of marine resources. It has been reported that aligate has many biological and physiological functions in human and has many appilcations in parmarceutical, food and agricultural industries. Therefore, much attention has been paid to the alginate and its hydrolysis products. Especially oligosaccharides from alginate by chemical and enzymatic hydrolysis has more biological activity and a new functions. However, the chemical hydrolysis process has many drawbacks. Therefore, many researchers have been interested in alginate hydrolysis by using alginate lyase from different sources, especially from microorganisms. Microbial alginate lyase has very high activity, different alginate lyase from different microorganisms has different substrate specificity and microbial alginate lyase can be easily produced by fermentattion.It has been reported that the marine bacterium Pseudoalteromonas elyakovii which was isolated from a decaying thallus of Laminaria is a pathogen of Laminaria. Interestingly, the extracellular alginate lyase produced by Pseudoalteromonas elyakovii is capable of degrading all block structures derived from sodium alginate and produces a series of tri to octaoligouronates. This alginate lyase is novel in terms of its broad substrate specificity. In addition to the metabolic studies, the enzymes from P. elyakovii have been used successfully to produce oligosaccharides with properties that are particularly useful in the preparation of specific food products (Sawabe et al. 2001). Therefore, it is very important to produce a large amount of the extracellular alginate lyase by overexpression in engineered E. coli in order to facilitate the production of alginate oligosaccharides for applications in the food industry. It is very important to understand its secretion and processing in the engineered E.coli in order to enhance the recombinant alginate lyase production. The main aims of the present study are to overexpress alyPEEC from P. elyakovii in E. coli and purify and characterize the recombinant alginate lyase. Hydrolysis of sodium alginate, polyM, and polyG by the purified recombinant alginate lyase was also carried out in this study. Another aims of the study are to elucidate the mechanisms of secretion and processing of the recombinant alginate lyase in E. coli.alyPEEC gene encoding alginate lyase in P. elyakovii has been cloned and sequenced. The whole ORF encodes a preproprotein of 398 amino acids which contains three domains : a signal peptide, N-terminal propeptide and the mature protein region. During secretion, it will be processed to produce the mature alginate lyase by removing the a signal peptide and N-terminal propeptide and the molecular mass of the mature alginate lyase is 32 kDa .In order to study the processing and secretion of alginate lyase in E. coli, we first purified the alginate lyase from the wild type Pseudoalteromonas elyakovii IAM 14594. The polyclonal antibody against alginate lyase was obtained by immunizing the mice with the purified alginate lyase. The results show that indirect antibody titer is 1 / 128 by ELISA and the polyclonal antibody can specificialy react with the purified alginate lyase.In this study, the alyPEEC gene encoding alginate lyase from marine bacterium Pseudoalteromonas elyakovii IAM 14594 was subcloned into pBAD24 with arabinose promoter and sequenced, and overexpressed in TOP10 strain of E. coli after arabinose induction. Two recombinant expression vectors: pBAD-ALY which contined the whole ORF encoding a signal peptide, N-terminal propeptide and the mature protein region of alginate lyase and pBAD-csALY which only contained the gene encoding the mature protein region of alginate lyase were constructed. Expression levels of alyPEEC gene in E. coli cells were over 7.5-fold higher than those in P. elyakovii IAM 14594 cells. It was also found that production of the recombinntant alginate lyase was greatly enhanced when the engineered E. coli TOP10 was grown in LB medium prepared with seawater. NaCl, MgCl2 and CaSO4 in the artificial seawater were found to play an important role in the enhancement of production of the recombinntant alginate lyase. After the amount of mRNA encoding alginate lyase was analyzed by RT-PCR in both E. coli cells grown in LB medium prepared with seawater and distiled water, we found that the enhancement of production of the recombinntant alginate lyase did not happened at the trascriptional levels. It is very interesting to observe that the enhancement of production of the recombinntant alginate lyase was due to enhancement of production of the mature aginate lyase after the specific protein bands in cytoplasmic, periplasmic and supernatant fractions was measured by western blotting.Because sec and tat mutants derived from the TOP10 strain are unavailable, we analyzed translocation of the recombined alginate lyase in CU164A (secY), B1LK0A (tatC) and their parental wild type strain MC4100A. Given that SecY is essential for bacterial viability, cold-sensitive secY mutant was used in this study. The Tat system is not essential for E. coli growth in LB media. The mutant B1LK0A has no functional Tat system since the essential tatC gene is completely deleted from its genome. The recombinant expression vector pBAD-ALY was transformed into MC4100A (wild typet), CU164A (secY mutant) and BILKOA (the tatC mutant) of E. coli, and expression of AlyPEEC was induced by adding arabiose. Protein samples were prepared, resolved on SDS-denaturing gels and analyzed by immunoblot using anti-alginate lyase antibodies. The results showed that the recombinant alginate lyase is exported into the periplasm via the Sec pathway in E. coli.Purified alginate lyase from the engineered E. coli cells was monomeric enzyme with molecular mass of 32 kDa. Optimum pH and temperature of the alginate lyase activity were 7.5 and 30oC, respectively. The enzyme was unstable on heating and in acidic and alkaline solution. The enzyme activity was stimulated by the MgCl2, NaCl, KCl, CaCl2, BaCl2 and MnCl2, but was inhibited by the addition of 1.0 mM of EGTA, EDTA, SDS, ZnSO4, AgNO3, CoCl2 and PMSF. All the alginate, polyM and polyG could be converted into penta-hexasaccharides by the purified recombinant alginate lyase, suggesting that the recombinant alginate lyase produced by the engineered E. coli has highly potential application in seaweed genetics, food and pharmaceutical industries.