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Improvement of optimum pH and specific activity of pectate lyase from Bacillus RN.1 using loop replacement.

ABSTRACT: Background: Alkaline pectate lyase plays an important role in papermaking, biological refining and wastewater treatment, but its industrial applications are largely limited owing to its low activity and poor alkali resistance. Methods: The alkaline pectate lyase BspPel from Bacillus RN.1 was heterologously expressed in Escherichia coli BL21 (DE3) and its activity and alkali resistance were improved by loop replacement. Simultaneously, the effect of R260 on enzyme alkaline tolerance was also explored. Results: Recombinant pectate lyase (BspPel-th) showed the highest activity at 60°C and pH 11.0, and showed significant stability over a wide pH range (3.0-11.0). The specific enzyme activity after purification was 139.4 U/mg, which was 4.4 times higher than that of the wild-type enzyme. BspPel-th has good affinity for apple pectin, since the V _max and K _m were 29 μmol/min. mL and 0.46 mol/L, respectively. Molecular dynamics simulation results showed that the flexibility of the loop region of BspPel-th was improved. Conclusion: The modified BspPel-th has considerable potential for industrial applications with high pH processes.

SUBMITTER: Li P

PROVIDER: S-EPMC10352616 | biostudies-literature | 2023

REPOSITORIES: biostudies-literature

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Improvement of optimum pH and specific activity of pectate lyase from Bacillus RN.1 using loop replacement.

Li Piwu P Wei Xiaofeng X Wang Yun Y Liu Hui H Xu Yanpeng Y Zhang Ziyang Z Li Junlin J Wang Jianbin J Guo Chuanzhuang C Sui Songsen S Wang Junqing J Wang Ruiming R

Frontiers in bioengineering and biotechnology 20230704

Background: Alkaline pectate lyase plays an important role in papermaking, biological refining and wastewater treatment, but its industrial applications are largely limited owing to its low activity and poor alkali resistance. Methods: The alkaline pectate lyase BspPel from Bacillus RN.1 was heterologously expressed in Escherichia coli BL21 (DE3) and its activity and alkali resistance were improved by loop replacement. Simultaneously, the effect of R260 on enzyme alka ...[more]

PMID: 37469444

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Project description:BackgroundPollen released by allergenic members of the botanically unrelated families of Asteraceae and Cupressaceae represent potent elicitors of respiratory allergies in regions where these plants are present. As main allergen sources the Asteraceae species ragweed and mugwort, as well as the Cupressaceae species, cypress, mountain cedar, and Japanese cedar have been identified. The major allergens of all species belong to the pectate lyase enzyme family. Thus, we thought to investigate cross-reactivity pattern as well as sensitization capacities of pectate lyase pollen allergens in cohorts from distinct geographic regions.MethodsThe clinically relevant pectate lyase pollen allergens Amb a 1, Art v 6, Cup a 1, Jun a 1, and Cry j 1 were purified from aqueous pollen extracts, and patients' sensitization pattern of cohorts from Austria, Canada, Italy, and Japan were determined by IgE ELISA and cross-inhibition experiments. Moreover, we performed microarray experiments and established a mouse model of sensitization.ResultsIn ELISA and ELISA inhibition experiments specific sensitization pattern were discovered for each geographic region, which reflected the natural allergen exposure of the patients. We found significant cross-reactivity within Asteraceae and Cupressaceae pectate lyase pollen allergens, which was however limited between the orders. Animal experiments showed that immunization with Asteraceae allergens mainly induced antibodies reactive within the order, the same was observed for the Cupressaceae allergens. Cross-reactivity between orders was minimal. Moreover, Amb a 1, Art v 6, and Cry j 1 showed in general higher immunogenicity.ConclusionWe could cluster pectate lyase allergens in four categories, Amb a 1, Art v 6, Cup a 1/Jun a 1, and Cry j 1, respectively, at which each category has the potential to sensitize predisposed individuals. The sensitization pattern of different cohorts correlated with pollen exposure, which should be considered for future allergy diagnosis and therapy.

Dataset Information

Improvement of optimum pH and specific activity of pectate lyase from Bacillus RN.1 using loop replacement.

Publications

Improvement of optimum pH and specific activity of pectate lyase from <i>Bacillus</i> RN.1 using loop replacement.

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