Studies On Enzymatic Chemistry, Product Analysis And Applications Of Alginate Lyase System

Alginate lyase is an algal tool enzyme. The studies and applications of alginate lyase have been an important development area of biofunctional resources from marine organisms. Alginate lyase system consists of poly-β-D-mannuronate lyase and poly-β-L-guluronate lyase according to their different substrate specificities. The studies on enzymatic chemistry, product analysis and applications were first carried out using the high-yield strain HZJ216 of alginate lyase that is owned by our lab. Using the crystal structure of the alginate lyase A1-â…¡', the interactions of the lyase and its substrates under the background of various solvents were explored through molecular dynamics simulation method. This is the first time to study on alginate lyase system produced from marine microorganism resources. The primary results are shown as below:The strain used in the lab showed high similarity with Pseudomonas fluorescens through molecular, physical and biochemical methods and was named as Pseudomonas fluorescens HZJ216. The enzymatic activity of the supernatant of its zymotic fluid was 975 EU mL^-1 measured by DNS method. The enzymatic system consisted of three alginate lyases with 60.25, 36, 23 kDa (named as the alginate lyases A, B and C, respectively) was purified. Their isoelectric points were 4, 4.36, and 4.59 through isoelectric focusing electrophoresis. The sequencing for N-termini of the three enzymes demonstrated that the alginate A and C are novel ones. Their optimal pH and temperature were 7.0 and 35?C, respectively. Their thermostabilization reduced with temperature increase and the enzyme A showed higher thermostabilization compared with the enzyme B. Their enzymatic activities were increased with Na⁺, K⁺, and Mg²⁺ to some extent. The enzyme A showed degradation ability on both pM and pG, however, the enzyme B and C were only specific to pM and pG, respectively. The results demonstrated different substrate specificities of the alginate lyase system. Structure-activity relationship and catalytic mechanism of alginate lyase were studied preliminarily. The comparative study on the open structure (2CWS) and close structure (2Z42) consisted of the loop L1 and L2 demonstrated that the relative movement of the loop L1 to L2 represents the off/on status and L1 is essential in the regulation of enzymatic activity. A newly identified loop L3 was found to play a possibly important role in substrate recognition. The molecular dynamics simulations of the region 2CWS illustrated that the change in energetic value is 7.08 kcal/mol during the shift from close to open status of enzymatic activity center while the energetic value of a hydrogen bond is usually 3-7 kcal/mol, indicating that hydrogen bond is crucial for maintaining the close status of the A1-â…¡'and its inactivation of alginate lyase.Furthermore, the intermediate transition structure of the enzymatic reaction was attained and Tyr284 was proved to function to catalyze the acidic step during the reaction through molecular dynamics simulations of the complex of the A1-â…¡'region of alginate lyase and its substrates.The sodium alginate was digested by the enzymatic system from the strain HZJ and the product was then precipitated by adding HCl and ethanol, two types of final products were obtained and six unsaturated oligosaccharides were purified from them by anion-exchange chromatography. Their structures were identified as DM, DMM, and DGM in which the reducing terminal ends with mannuronate and DG, DGG, and DMG in which the reducing terminal ends with guluronate through structure analysis by ESI-MS and 2DNMR. This is the first time to obtain all kinds of unsaturated disaccharides and trisaccharides. The results also demonstrated that the lyases consisted of alginate lyase system has different substrate specificities.The alginate lyase system was employed to prepare enzyme preparation because of its stable and high activity. The solid enzyme preparation showed 60,000U/g enzyme activity prepared by both salt-out method and acetone precipitation method from ferment fluid that was 975EU mL^-1 activity and 3034 U mg^-1 specific activity. The solid enzyme prepared by salt-out method kept 90% activity and the ones prepared by acetone precipitation kept 85% activity when they were stored at ?20?C for 12 months, respectively. The test results, such as heavy metal, Salmonella, total plate count and Escherichia coli, demonstrated that the two enzyme preparations correspond the general quality standard of FAO and WHO for enzyme preparation products.Alginate lyase can be used as tool enzyme to prepare low viscosity and ultra-low viscosity alginate. Low viscosity alginate (100～200 mPa·s) was prepared after adding 0.1-0.75 mL (975 EU mL^-1) enzyme solution to 300 mL digestive solution made from 10 g dried seaweed. Its yield improved by 1-7% and water saving ratio improved by 10-40% compared with the traditional process. When 0.75～2 mL (975 EU mL^-1) enzyme solution was added, ultra-low viscosity alginate (10-100 mPa·s) was prepared. Its yield improved by 7-15% and water save ratio reached 40-50% compared with the traditional process.Using the strain that produces alginate lyase system stably and efficiently serial researches were performed on strain classification, enzyme characteristics, analysis of enzyme-hydrolytic end products, and enzyme applications. The results demonstrated the different substrate specificities of the lyases in the lyase system. We obtained all kinds of alginate disaccharides and trisaccharides, prepared high-efficient and stable enzyme preparation, summarized an enzyme controlled process for preparing new type alginate using the enzyme system in this study. The catalytic mechanism of this type of lyase was for the first time approached by molecular dynamics simulation. These studies provide important theoretical basis and application value for application and development of novel marine tool enzymes and alginate products.