Optimization of Culturing Condition and Medium Composition for the Production of Alginate Lyase by a Marine Vibrio sp. YKW-34

Carbohydrases secreted by marine Vibrio sp. YKW-34 with strong Laminaria cell wall degrading ability were screened, and among them alginate lyase was found to be dominant. The effects of medium composition and culturing condition on the produc- tion of alginate lyase by marine Vibrio sp. YKW-34 in flask were investigated in this study. In the culture medium of marine broth, no alginate lyase was produced. The activity of the alginate lyase, after being induced, reached 5 UmL–1. The best inoculum volume and inoculum age were 10% and 12 h, respectively. The optimal temperature for alginate lyase production was 25℃. The fermentation medium was composed of 0.5% of Laminaria powder and 0.2% of KNO3 with an initial acidity of pH 8.0. Alginate could induce alginate lyase production but not as efficiently as Laminaria powder did. The addition of fucoidan, cellulose and glucose had nega- tive effect on the alginate lyase production. Other kinds of nitrogen sources, such as yeast extract, beef extract and peptone, had posi- tive effect on the growth of the microorganism and negative effect on alginate lyase production. In addition, the time course of algi- nate lyase production under the optimized condition was described. The optimal harvest time was 48 h.

Expression and characterization of a bifunctional alginate lyase named Al163 from the Antarctic bacterium Pseudoalteromonas sp. NJ-21

In this study, an endolytic alginate lyase, named Al163, was identified, cloned, and characterized from the Antarctic bacterium Pseudoalteromonas sp. NJ-21. Comparative sequence analysis showed that the predicted amino acid sequence encoded by al163 belongs to the polysaccharide lyase 6(PL-6) family and has a molecular mass of about 80 kDa. Recombinant enzyme was purified by Ni-Sepharose affinity chromatography. Recombinant Al163 exhibited maximum activity(258 U/mg) at pH 7.0 and 40℃, and thermal stability assays showed retention of almost 90% activity after incubation at 30℃ for 30 min. Al163 activity was stimulated by Cd^(2+), Ca^(2+), Fe^(3+), and Mn^(2+), but inhibited by Cu^(2+), Si^(2+), Fe^(2+), and Ni^(2+). Thin-layer chromatographic analysis indicated that Al163 degraded sodium alginate, poly M, and poly G, generating disaccharides and trisaccharides as the final products. Only a few bacterial strains that produce a bifunctional alginate lyase have been reported. Our results indicate that recombinant Al163 exhibits broad substrate specificity and its products exhibit low degrees of polymerization. Both properties imply high potential for use of the enzyme in several industrial fields, including cosmetics and pharmaceuticals, based on the high demand for biologically active oligosaccharides.

Isolation of Protoplasts from Undaria pinnatifida by Alginate Lyase Digestion

The aim of this study is to isolate protoplasts from Undaria pinnatifida. Protoplasts of the alga were isolated enzymatically by using alginate lyase, which was prepared by fermenting culture of a strain Vibrio sp. 510. Monofacterial method was applied for optimizing digestion condition. The optimum condition for protoplast preparation is enzymatic digestion at 28 ℃ for 2 h using alginate lyase at the concentration of 213.36 U (8 mL) every 0.5 g fresh thalline with NaCl 50 and at the shaking speed of 150 r min -1 during digestion. The protoplast yield can reach 2.62±0.09 million per 0.5 g fresh leave under the optimum condition. The enzyme activity is inhibited by Ca 2+ and slightly enhanced by Fe 2+ and Mn 2+ at concentrations of 0.05, 0.08 and 0.10 mol L -1.

Evolving strategies for marine enzyme engineering:recent advances on the molecular modification of alginate lyase

Alginate,an acidic polysaccharide,is formed byβ-D-mannuronate(M)andα-L-guluronate(G).As a type of polysaccharide lyase,alginate lyase can efficiently degrade alginate into alginate oligosaccharides,having potential applications in the food,medicine,and agriculture fields.However,the application of alginate lyase has been limited due to its low catalytic efficiency and poor temperature stability.In recent years,various structural features of alginate lyase have been determined,resulting in modification strategies that can increase the applicability of alginate lyase,making it important to summarize and discuss the current evidence.In this review,we summarized the structural features and catalytic mechanisms of alginate lyase.Molecular modification strategies,such as rational design,directed evolution,conserved domain recombination,and non-catalytic domain truncation,are also described in detail.Lastly,the application of alginate lyase is discussed.This comprehensive summary can inform future applications of alginate lyases.

Cloning,expression and improvement of catalytic activity of alginate lyase by site-directed mutation

Alginate lyase mainly produces active alginate oligosaccharides(AOS)by degrading alginate viaβ-elimination process.In this study,the Pseudoalteromonas sp.Alg6B alginate lyase-encoding gene alg6B-7 from polysaccharide lyase(PL)-7 family was successfully cloned,sequenced,expressed in Escherichia coli.Based on rational design and amino acid sequence alignment of the alginate lyase from various sources,four positive mutants were obtained.The specific enzyme activities of four mutants I62A,A99K,V132S,and L157T were 38.84%,42.85%,75.8%and 51.83%higher than that of the wild enzyme,respectively.The K_(cat)/K_(m) values of the four mutants were both increased,and the catalytic efficiency of V132S was 1.92-fold higher than that of the wild enzyme,especially.The rational design that was employed in this study achieved the dramatic improvement of catalytic activity,which may provide the application potential in industrial production.

Enzymatically prepared alginate oligosaccharides improve broiler chicken growth performance by modulating the gut microbiota and growth hormone signals

Background Alginate oligosaccharide(AOS)holds great potential as a novel feed supplement in farm animals.However,the effects of AOS on chicken health and the underlying mechanisms are not fully understood.This study aimed to optimize the enzymatic preparation of AOS by using bacterial alginate lyases expressed in yeast,investigate the effects of the prepared AOS on the growth performance and gut health of broiler chickens,and reveal the underlying mechanisms.Results Five alginate lyases from bacteria were cloned into Pichia pastoris GS115 and the alginate lyase PDE9 was expressed at relatively high yield,activity and stability in P.pastoris.Animal trials were carried out using 3201-day-old male Arbor Acres broilers(four groups;8 replicates/group×10 chicks/replicate)receiving either a basal diet or the same diet supplemented with 100,200 and 400 mg/kg PDE9-prepared AOS for 42 d.The results showed that dietary supplementation of 200 mg/kg AOS displayed the highest activity in promoting the birds’ADG and ADFI(P<0.05).AOS ameliorated the intestinal morphology,absorption function and barrier function,as indicated by the enhanced(P<0.05)intestinal villus height,maltase activity,and the expression of PEPT,SGLT1,ZNT1,and occludin.AOS also increased serum insulin-like growth factor-1,ghrelin(P<0.05),and growth hormone(P<0.1).Moreover,the concentrations of acetate,isobutyrate,isovalerate,valerate,and total SCFAs in cecum of birds fed AOS were significantly higher than the control birds(P<0.05).Metagenomic analysis indicated that AOS modulated the chicken gut microbiota structure,function,and microbial interactions and promoted the growth of SCFAs-producing bacteria,for example,Dorea sp.002160985;SCFAs,especially acetate,were found positively correlated with the chicken growth performance and growth-related hormone signals(P<0.05).We further verified that AOS can be utilized by Dorea sp.to grow and to produce acetate in vitro.Conclusions We demonstrated that the enzymatically produced AOS effectively promoted broiler chicken growth performance by modulating the chicken gut microbiota structure and function.For the first time,we established the connections among AOS,chicken gut microbiota/SCFAs,growth hormone signals and chicken growth performance.

P_(luxI)mutants with different promoting period and their application for quorum sensing regulated protein expression

Quorum sensing(QS)system can dynamically control the expression of proteins along with the cell growth.The promoting period of QS system has been little focused on until now.In this study,a self-induced dynamic regulated expression(SIDRE)system was constructed in Escherichia coli.To enable the system suitable for the expression of enzymes,promoter engineering was used to obtain P_(luxI)mutants.To test the SIDRE system,alginate lyase AL493 and esterase Est7 were used as target protein for expression.The enzyme activity of alginate lyase and esterase reached 96.38%and 106.71%of the control strains containing the T7 promoter.In high-density fermentation,the activity of alginate lyase expressed by the SIDRE system with P_(luxI)(T-38C)as promoter was 4.34-fold of that expressed by the T7 promoter.Therefore,the P_(luxI)mutants with different promoting periods and/or different strengths show great potential in both laboratory and industrial scale for protein expression.