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Mar Drugs
2022 Nov 29;2012:. doi: 10.3390/md20120753.
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Insights into the Influence of Signal Peptide on the Enzymatic Properties of Alginate Lyase AlyI1 with Removal Effect on Pseudomonas aeruginosa Biofilm.
Zhang MJ
,
Yun ST
,
Wang XC
,
Peng LY
,
Dou C
,
Zhou YX
.
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Most reports on signal peptides focus on their ability to affect the normal folding of proteins, thereby affecting their secreted expression, while few studies on its effects on enzymatic properties were published. Therefore, biochemical characterization and comparison of alginate lyase rALYI1/rALYI1-1 (rALYI1: without signal peptides; rALYI1-1:with signal peptides) were conducted in our study, and the results showed that the signal peptide affected the biochemical properties, especially in temperature and pH. rALYI1 (32.15 kDa) belonging to polysaccharide lyase family 7 was cloned from sea-cucumber-gut bacterium Tamlana sp. I1. The optimum temperature of both rALYI1 and rALYI1-1 was 40 °C, but the former had a wider optimum temperature range and better thermal stability. The optimum pH of rALYI1 and rALYI1-1 were 7.6 and 8.6, respectively. The former was more stable and acid resistant. Noticeably, rALYI1 was a salt-activated enzyme and displayed remarkable salt tolerance. Alginate, an essential polysaccharide in algae and Pseudomonas aeruginosa biofilms, is composed of α-L-guluronate and β-D-mannuronate. It is also found in our study that rALYI1 is also effective in removing mature biofilms compared with controls. In conclusion, the signal peptide affects several biochemical properties of the enzyme, and alginate lyase rALYI1 may be an effective method for inhibiting biofilm formation of Pseudomonas aeruginosa.
31700116 National Natural Science Foundation of China, ZR2017MC019 Natural Science Foundation of Shandong Province, 2017M62218 China Postdoctoral Science Foundation, 1070413421511 The Key Science and Technology Program of Weihai
Figure 1. Phylogenetic analysis of AlyIl with other reported alginate lyases. The reliability of the phylogenetic reconstructions was determined by boot-strapping values (1000 replicates). Branch-related numbers are bootstrap values (confidence limits) representing the substitution frequency of each amino acid residue. The species names are indicated along with the accession number of the corresponding alginate lyase sequence. Bootstrap values of 1000 trials are presented in the branching points. Bar, 0.20 substitutions per nucleotide position.
Figure 2. Multiple sequence alignments of alginate lyase AlyI1 and related alginate lyases of the PL7 family. AlyI1 (ON186791) from Tamlana sp. I1 in this study, AlyA1-II (BAD16656) from Sphingomonas sp. A1, Alg2A(AEB69783) from Flavobacterium sp. S20, AlyA (AAA25049) from Klebsiella pneumonia subsp Aerogenes type 25, Aly7B_Wf (ANW96808.1) from Wenyingzhuangia fucanilytica, and alyVI (AAP45155) from Vibrio sp. QY101. The conserved amino acid regions are highlighted with red boxes. The potential residues involved in the catalytic activity in the PL7 family are indicated with green triangles. The depth of the color determines the degree of homology. Dark blue represented conserved amino acids. Pink and cyan represented amino acids with similar functions and structures, with pink representing a higher homology.
Figure 3. Peak bitmap of secondary structure. (a) The fitting results of amide I band of ALYI1. (b) The fitting results of the amide I band of ALYI1-1.
Figure 4. Biochemical characteristics of rALYI1/rALYI1-1. (a) Relative activity of rALYI1/rALYI1-1 at different temperatures (4–70 °C). (b) Thermostability of rALYI1/rALYI1-1. (c) The optimal pH of rALYI1/rALYI1-1. (d) pH stability of rALYI1/ rALYI1-1. (e) Effects of metal ions and surfactants. (f) Effects of NaCl on rALYI1/rALYI1-1. Data are shown as the means ± standard deviation, n = 3. * p < 0.1; ** p < 0.05.
Figure 5. Analysis substrate specificity and final products. (a) Substrate specificity of rALYI1/rALYI1-1. (b) TLC analysis of rALYI1. Lane 1–3, the purified monomeric sugar, dimer, and trimer standards; lane 4–6, the degradation products of alginate, PM, PG. (c) ESI-MS analysis of 24 h enzymatic hydrolysates of rALYI1 using alginate as substrate. The DP2 and DP3 peaks represent a disaccharide and trisaccharide, respectively. DP2 ([∆DP2–H]–: m/z 375), DP3 ([∆DP3+Na–2H]–: m/z 546), DP4 ([∆DP4+Na–2H]– : m/z 727), DP5 ([∆DP3+Na–2H]– : m/z 934), and DP6 ([∆DP6–H]–: m/z 1079). ** p < 0.05.
Figure 6. Effect of alginate lyase rALYI1 on biofilm produced by P. aeruginosa. (a) Biofilm disruptive activity of rALYI1. (b) Prevention of biofilm formation of rALYI1. ** p < 0.05. (c) Blank control of rAlyI1 inhibiting biofilm formation after crystal violet staining. (d) rALYI1 inhibits biofilm formation after crystal violet staining. (e) Blank control of rALYI1 destruction of biofilms after crystal violet staining. (f) rALYI1 destroys biofilms after crystal violet staining. The scale bar corresponds to 100 μm.
Figure 7. MTT viable bacteria count results. (a) Biofilm disruptive activity of rALYI1. (b) Prevention of biofilm formation of rALYI1.
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