| Acid mine drainage（AMD）is a typical extremely acidic environment characterized by extremely low p H,oligotrophic and rich in heavy metals.Microorganisms in this habitat have special metabolic pathways and adaptive mechanisms,which have unique advantages in bioresource exploitation.However,only a few acidophilic microorganisms have been isolated,and a substantial number of non-culturable microorganisms in AMD and their genetic resources have not been fully explored.β-mannanase（EC 3.2.1.78）is a class of hemicellulases that act on theβ-1,4-linked glycosidic bond to degrade mannans into oligosaccharides with various functional activities,and are widely used in many fields such as feed and food industries.β-mannanases are distributed in four glycoside hydrolysis（GH）families,but the number of GH113 family members is limited and a comprehensive understanding of the biochemical properties and molecular mechanisms of this family is lacking.Moreover,GH113β-mannanases are poorly applied in feed industries and juice processing due to its unsatisfactory acid tolerance properties.Based on this,in this paper,two novel GH113 familyβ-mannanases were identified from AMD using a metagenome approach,and their enzymatic properties were investigated;The acid tolerance and catalytic activity of the novelβ-mannanase Man113Ali9 were improved by using directed evolution and semi-rational design techniques,and the relationship between their acid tolerance and protein structure was elucidated at the molecular level.This study will provide a theoretical basis for the modification of acid/base properties and functional enhancement of related glycoside hydrolases.The specific results of the study are as follows:（1）Acid mine drainage samples were collected from Dabaoshan Mine,Guangdong,and enriched with konjac flour as carbon source for the enrichment of mannan-degrading microorganisms,and metagenome sequencing was performed on the original and enriched samples.During the enrichment process,the abundance of Acidiphilium sp.increased significantly,and the proportions of Acidobacterium sp.and Alicyclobacillus sp.also increased.The AMD metagenome contained a rich diversity of carbohydrate-active enzymes genes,and the abundance of mannan-degrading enzymes genes increased significantly in the enriched samples.（2）Based on the constructed hidden Markov models specific for different families ofβ-mannanases,25 potentialβ-mannanase genes were annotated from the AMD metagenome.Seven of them were cloned,and man113Ali8 and man113Ali9 genes were expressed in Escherichia coli solublely.Man113Ali8 and Man113Ali9 belonged to the GH113 family,and exhibited the highest identity（68%）to the reported GH113β-mannanase;Man113Ali8 and Man113Ali9 displayed optimal activity at p H6.0 and 50℃,with over 75%activity retained at p H 5.0-9.0,and good thermal stability at 50℃;The addition of Tween 80 enhanced enzyme activity.Man113Ali8 and Man113Ali9 were capable to hydrolyze various mannan substrates,producing hydrolysis products dominated with mannobiose and mannotetraose,respectively.Man113Ali8 and Man113Ali9 also appeared to exhibit transglycosylation activity toward mannotetraose to mannohexaose.（3）A random mutant library of the enzyme Man113Ali9 was constructed using a combination of error-prone PCR and DNA shuffling.A positive mutant m Man113Ali9 was obtained by high-throughput screening in 96-well plates,and the mutant retained 53%of the enzyme activity after 1-h incubation at p H 4.0,while its specific activity at p H 5.0was 2.6 times that of the wild enzyme.The m Man113Ali9 mutant had four mutation sites（Y97F,N119S,V246M,F252Y）,of which the residues Tyr252 and Met246 were the key sites which cause the change in enzyme properties.The introduction of a new hydrogen bond at Tyr252and a new van der Waals force at Met246 may be associated with improved acid stability;Molecular dynamics simulations showed that the mutant structure was less displaced and the protein structure was tighter in the low p H condition,and Met246reduced the fluctuation of the neighboring loop region（No.238-243 residues）by stabilizing theα-helix where it was located.（4）We used amino acid sequences alignment and high net charge design strategy to construct several single point mutants,from which a mutant Y236W with enhanced acid stability was obtained.The mutant enzyme with combined substitution（Y236W/V246M/F252Y）showed the most pronounced shifts in acid tolerance,which retained 68%of the enzyme activity after 1-h treatment at p H 4.0,showed 2.7 times higher specific activity than the wild enzyme at p H 5.0,increased the optimum temperature by 10°C and improved the thermal stability at 60℃.The combined mutation did not affect the composition of the hydrolysis profile of mannans.Substitution of site 236 for Trp enhanced the bond energy ofπ-πstacking with His174,changed the shape of the substrate binding pocket and hydrophobic forces,and the superimposed hydrogen bonding and van der Waals forces introduced at sites 252 and 246 further enhanced the structural stability of the enzyme,thus improving the acid/thermal properties of the mutant.Molecular dynamics simulations also confirmed the more stable structure of the combined mutant under acidic condition. |
