Alginate lyases

Sources, structure- gene composition , Production- extraction & purification of Alginate Lyase,Types- Alginate I, Alginase Lyase II, Uses- Drugs, Food additives, Compost

1. ALGINATE LYASES M.MEENAKSHI, ASSISTANT PROFESSOR, DEPARTMENT OF MICROBIOLOGY, SRI RAMAKRISHNA COLLEGE OF ARTS & SCIENCE, COIMBATORE

2. INTRODUCTION The enzymes have been isolated from various kinds of organisms with different substrate specificities, including algae, marine mollusks , marine and terrestrial bacteria, and some viruses and fungi

3. STRUCTURE

4. PRODUCTION OF ALGINATE

5. PRODUCTION OF ALGINATE LYASE

6. FERMENTATION PROCESS A high-density-cell fermentation process for production of an exracellular alginate lyase from  Klebseilla pneumoniae  on a defined medium has been developed. The process employs a strategy using two carbon sources . One low-molecular-mass, low-viscosity carbon source (sucrose) with high water solubililty is used as the main carbons source for growth, while the high-molecular-mass and viscous alginate in low concentration is used as an inducer for enzyme synthesis.  The repression of alginate lyase production by sucrose and the growth inhibition that we observed at increased levels of ammonia were circumvented by a computer-assisted fed-batch addition of the carbon sources ( succrose and alginate) and by supplying nitrogen source as ammonia in the pH control. 

7. FERMENTATION PROCESS…… No enzyme production was observed when dissolved oxygen limited growth at an oxygen uptake rate of 40%–50% of the maximum uptake rate. An optimal composition of the feeding solution (12.5 g alginate and 587.5 g sucrose 1 −1 ) was found both for the maximum final concentration of enzyme (1330 U 1 −1 ) and for the maximum volumetric rate of enzyme production (67 U 1 −1  h −1 ). The enzyme production depends of the growth rate in the linear growth phase, giving a maximum enzyme concentration at the highest growth rate tested.  The final enzyme concentration shows a fivefold increase compare with previously reported data where alginate was used as a carbon source. In addition, the ratio of alginate lyase by a factor of apporximately 15. A doubling in extracellular specific activity of the enzyme was observed, a property of significant interest, especially for purification of the enzyme. On the other hand, the final dry cell weight concentration of the bacteria also increased by a factor of 15–20 thus giving a relatively lower specific productivity of 0.4 U (g cell dry weight) −1  h −1 .

8. ALGINATE LYASES Alginate lyases  are group of  enzymes  which catalyze depolymerization of  alginate  into oligosaccharides.  Alginate lyase  have been widely used in many applications such as in production of bioactive oligosaccharides, control of polysaccharide rheological properties, and polysaccharide structure analysis . This enzyme belongs to the family of  lyases , specifically those carbon-oxygen lyases acting on polysaccharides. The  systematic name  of this enzyme class is  poly(beta-D-1,4-mannuronide) lyase . Other names in common use include  alginate lyase I ,  alginate lyase ,  alginase I ,  alginase II , and  alginase . This enzyme participates in  fructose  and  mannose metabolism .

9. SOURCE AND CLASSIFICATION Alginate lyases have been isolated from various sources, including marine algae, marine mollusks ( Littorina spp .,  Haliotis spp ,  Turbo cornutus .), and a wide range of marine and terrestrial bacteria. In addition, some lyases have been isolated from fungi and viruses . The alginate lyases can be classified into 2 groups due to their substrate specificities, one is G block-specific lyase ( polyG lyase , EC4.2.2.11), and the other is M block-specific lyase ( polyM lyase , EC4.2.2.3). This classification has been widely accepted, but some enzymes show activities toward both polyM and polyG, 30-33  which may degrade alginate more effectively. In terms of the mode of action, alginate lyase can be grouped into endolytic and exolytic alginate lyase

10. SOURCE AND CLASSIFICATION In terms of the mode of action, alginate lyase can be grouped into endolytic and exolytic alginate lyase .   Endolytic alginate lyase cleaves glycosidic bonds inside alginate polymer and releases unsaturated oligosaccharides (di-, tri-, and tetra-saccharides) as main products,  while exolytic alginate lyase can further degrade oligosaccharides into monomers.   Based on the analysis of hydrophobic cluster of primary structures, alginate lyases can be grouped into 7 families of Polysaccharide Lyase (PL) family ( Table 2 ), PL-5, -6, -7, -14,-15, -17, and - 18.

11. SOURCE AND CLASSIFICATION Most of endolytic bacterial alginate lyases are assigned to PL-5 and PL-7. The most exolytic alginate lyases are grouped into PL-15 and PL-17 families. Most alginate lyases from bacteria are assigned into PL-5, -7, -15, and -17 families. The lyases isolated from marine mollusks and viruses are collected in PL-14 family. The bifunctional alginate lyases belong to PL-18 family, while other lyases are dispersed in other 6 families.  T he alginate lyases can also be grouped into 3 types based on their molecular masses: small (25–30  kDa ), mediu655m-sized (around 40  kDa ), and large lyases (>60  kDa ).

12. USES Pseudomonas aeruginosa  ( P. aeruginosa ) infections lead to a high mortality rate for cystic fibrosis or immunocompromised patients. The alginate of the biofilm was believed to be the key factor disabling immune therapy and antibiotic treatments. A silver nanocomposite consisting of silver nanoparticles and a mesoporous organosilica layer was created to deliver two pharmaceutical compounds (alginate lyase and ceftazidime) to degrade the alginate and eradicate  P. aeruginosa  from the lungs . The introduction of thioether -bridged mesoporous organosilica into the nanocomposites greatly benefited the conjunction of foreign functional molecules such as alginate lyase and increased their hemocompatibility and drug-loading capacity.

13. USES…. Silver nanocomposites with a uniform diameter (∼39 nm) exhibited a high dispersity, good biocompatibility, and high ceftazidime-loading capacity (380.96 mg/g ). Notably, the silver nanocomposites displayed a low pH-dependent drug release and degradation profiles (pH 6.4), guaranteeing the targeted release of the drugs in the acidic niches of the  P. aeruginosa  biofilm. Indeed , particles loaded with alginate lyase and ceftazidime exhibited high inhibitory and degradation effects on the biofilm of  P. aeruginosa  PAO1 based on the specific catalytic activity of the enzyme to the alginate and antibacterial function of their loaded ceftazidime and silver ions . It should be noted that the enzyme-decorated nanocomposites succeeded in eradicating  P. aeruginosa  PAO1 from the mouse lungs and decreasing the lung injuries . No deaths or serious side effects were observed during the experiments . We believe that the silver nanocomposites with high biocompatibility and organic group-incorporated framework have the potential to be used to deliver multiple functional molecules for antibacterial therapy in clinical application.

14. USES Alginate is mainly used as a food additive to modify food texture due to its high viscosity and gelling property. Alginate lyase can degrade alginate by cleaving the glycosidic bond through a β-elimination reaction, generating oligomer with 4-deoxy-L-erythro-hex-4-enepyranosyluronate at the nonreducing end . Alginate oligosaccharides have been shown to stimulate the growth of human endothelial cells and the secretion of cytotoxic cytokines from human macrophage. Alginate can be converted into unsaturated monosaccharide by saccharification process using endolytic and exolytic alginate lyases , thus alginate lyases have potential as key biocatalyst for application of alginate as a renewable source for biochemicals and biofuels in near future.

15. REFERENCES Aasen IM, Folkvord K, Levine DW (1992) Develpment of a process for large-scale chromatography purification of an alginate from  Klebsiella pneumoniae . Appl Microbiol Biotechnol 37:163–171 Boyd J, Turvey JR (1977) Isolation of a poly- α- L - guluronate lyase from  Klebsiella aerogenes . Carbohyr Res 57:163–171 Bod J, Turvey JR (1978) Structural studies of alginic acid, using a bacterial poly- α- L - guluronate lyase . Carbohydr Res 66:187–194 Boyen C, Bertheau Y, Barbeyron T, Kloareg B (1990a) Preparation of guluronate lyase from  Pseudomonas alginovora  for protoplast isolation in  Laminaria . Enzyme Micrbo Technol 12:885–890