scholarly journals Activation Mechanism and Cellular Localization of Membrane-Anchored Alginate Polymerase in Pseudomonas aeruginosa

2017 ◽  
Vol 83 (9) ◽  
Author(s):  
M. Fata Moradali ◽  
Shirin Ghods ◽  
Bernd H. A. Rehm
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Amino Acid ◽  
Structural Model ◽  
Second Messenger ◽  
Catalytic Site ◽  
Activation Mechanism ◽  
Amino Acid Residues ◽  
Human Pathogen ◽  
Content Type ◽  
Opportunistic Human Pathogen

ABSTRACT The exopolysaccharide alginate, produced by the opportunistic human pathogen Pseudomonas aeruginosa, confers a survival advantage to the bacterium by contributing to the formation of characteristic biofilms during infection. Membrane-anchored proteins Alg8 (catalytic subunit) and Alg44 (copolymerase) constitute the alginate polymerase that is being activated by the second messenger molecule bis-(3′, 5′)-cyclic dimeric GMP (c-di-GMP), but the mechanism of activation remains elusive. To shed light on the c-di-GMP-mediated activation of alginate polymerization in vivo, an in silico structural model of Alg8 fused to the c-di-GMP binding PilZ domain informed by the structure of cellulose synthase, BcsA, was developed. This structural model was probed by site-specific mutagenesis and different cellular levels of c-di-GMP. Results suggested that c-di-GMP-mediated activation of alginate polymerization involves amino acids residing at two loops, including H323 (loop A) and T457 and E460 (loop B), surrounding the catalytic site in the predicted model. The activities of the respective Alg8 variants suggested that c-di-GMP-mediated control of substrate access to the catalytic site of Alg8 is dissimilar to the known activation mechanism of BcsA. Alg8 variants responded differently to various c-di-GMP levels, while MucR imparted c-di-GMP for activation of alginate polymerase. Furthermore, we showed that Alg44 copolymerase constituted a stable dimer, with its periplasmic domains required for protein localization and alginate polymerization and modification. Superfolder green fluorescent protein (GFP) fusions of Alg8 and Alg44 showed a nonuniform, punctate, and patchy arrangement of both proteins surrounding the cell. Overall, this study provides insights into the c-di-GMP-mediated activation of alginate polymerization while assigning functional roles to Alg8 and Alg44, including their subcellular localization and distribution. IMPORTANCE The exopolysaccharide alginate is an important biofilm component of the opportunistic human pathogen P. aeruginosa and the principal cause of the mucoid phenotype that is the hallmark of chronic infections of cystic fibrosis patients. The production of alginate is mediated by interacting membrane proteins Alg8 and Alg44, while their activity is posttranslationally regulated by the second messenger c-di-GMP, a well-known regulator of the synthesis of a range of other exopolysaccharides in bacteria. This study provides new insights into the unknown activation mechanism of alginate polymerization by c-di-GMP. Experimental evidence that the activation of alginate polymerization requires the engagement of specific amino acid residues residing at the catalytic domain of Alg8 glycosyltransferase was obtained, and these residues are proposed to exert an allosteric effect on the PilZAlg44 domain upon c-di-GMP binding. This mechanism is dissimilar to the proposed mechanism of the autoinhibition of cellulose polymerization imposed by salt bridge formation between amino acid residues and released upon c-di-GMP binding, leading to activation of polymerization. On the other hand, conserved amino acid residues in the periplasmic domain of Alg44 were found to be involved in alginate polymerization as well as modification events, i.e., acetylation and epimerization. Due to the critical role of c-di-GMP in the regulation of many biological processes, particularly the motility-sessility switch and also the emergence of persisting mucoid phenotypes, these results aid to reach a better understanding of biofilm-associated regulatory networks and c-di-GMP signaling and might assist the development of inhibitory drugs.

scholarly journals Contextual Flexibility in Pseudomonas aeruginosa Central Carbon Metabolism during Growth in Single Carbon Sources

mBio ◽  
2020 ◽  
Vol 11 (2) ◽  
Author(s):  
Stephen K. Dolan ◽  
Michael Kohlstedt ◽  
Stephen Trigg ◽  
Pedro Vallejo Ramirez ◽  
Clemens F. Kaminski ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Metabolic Flux ◽  
Antimicrobial Agents ◽  
Carbon Sources ◽  
Aerobic Denitrification ◽  
Human Pathogen ◽  
Content Type ◽  
Opportunistic Human Pathogen

ABSTRACT Pseudomonas aeruginosa is an opportunistic human pathogen, particularly noted for causing infections in the lungs of people with cystic fibrosis (CF). Previous studies have shown that the gene expression profile of P. aeruginosa appears to converge toward a common metabolic program as the organism adapts to the CF airway environment. However, we still have only a limited understanding of how these transcriptional changes impact metabolic flux at the systems level. To address this, we analyzed the transcriptome, proteome, and fluxome of P. aeruginosa grown on glycerol or acetate. These carbon sources were chosen because they are the primary breakdown products of an airway surfactant, phosphatidylcholine, which is known to be a major carbon source for P. aeruginosa in CF airways. We show that the fluxes of carbon throughout central metabolism are radically different among carbon sources. For example, the newly recognized “EDEMP cycle” (which incorporates elements of the Entner-Doudoroff [ED] pathway, the Embden-Meyerhof-Parnas [EMP] pathway, and the pentose phosphate [PP] pathway) plays an important role in supplying NADPH during growth on glycerol. In contrast, the EDEMP cycle is attenuated during growth on acetate, and instead, NADPH is primarily supplied by the reaction catalyzed by isocitrate dehydrogenase(s). Perhaps more importantly, our proteomic and transcriptomic analyses revealed a global remodeling of gene expression during growth on the different carbon sources, with unanticipated impacts on aerobic denitrification, electron transport chain architecture, and the redox economy of the cell. Collectively, these data highlight the remarkable metabolic plasticity of P. aeruginosa; that plasticity allows the organism to seamlessly segue between different carbon sources, maximizing the energetic yield from each. IMPORTANCE Pseudomonas aeruginosa is an opportunistic human pathogen that is well known for causing infections in the airways of people with cystic fibrosis. Although it is clear that P. aeruginosa is metabolically well adapted to life in the CF lung, little is currently known about how the organism metabolizes the nutrients available in the airways. In this work, we used a combination of gene expression and isotope tracer (“fluxomic”) analyses to find out exactly where the input carbon goes during growth on two CF-relevant carbon sources, acetate and glycerol (derived from the breakdown of lung surfactant). We found that carbon is routed (“fluxed”) through very different pathways during growth on these substrates and that this is accompanied by an unexpected remodeling of the cell’s electron transfer pathways. Having access to this “blueprint” is important because the metabolism of P. aeruginosa is increasingly being recognized as a target for the development of much-needed antimicrobial agents.

scholarly journals Quorum Sensing Is Accompanied by Global Metabolic Changes in the Opportunistic Human Pathogen Pseudomonas aeruginosa

2015 ◽  
Vol 197 (12) ◽  
pp. 2072-2082 ◽  
Author(s):  
Peter W. Davenport ◽  
Julian L. Griffin ◽  
Martin Welch
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Fatty Acid ◽  
Quorum Sensing ◽  
Acid Methyl Ester ◽  
Metabolic Changes ◽  
Human Pathogen ◽  
Wild Type ◽  
Content Type ◽  
Ecological Fitness ◽  
Opportunistic Human Pathogen

ABSTRACTPseudomonas aeruginosausesN-acyl-homoserine lactone (AHL)-dependent quorum sensing (QS) systems to control the expression of secreted effectors. These effectors can be crucial to the ecological fitness of the bacterium, playing roles in nutrient acquisition, microbial competition, and virulence. In this study, we investigated the metabolic consequences of AHL-dependent QS by monitoring the metabolic profile(s) of alasI rhlIdouble mutant (unable to make QS signaling molecules) and its wild-type progenitor as they progressed through the growth curve. Analysis of culture supernatants by1H-nuclear magnetic resonance (1H-NMR) spectroscopy revealed that at the point where AHL concentrations peaked in the wild type, the metabolic footprints (i.e., extracellular metabolites) of the wild-type andlasI rhlImutant diverged. Subsequent gas chromatography-mass spectrometry (GC-MS)-based analysis of the intracellular metabolome revealed QS-dependent perturbations in around one-third of all identified metabolites, including altered concentrations of tricarboxylic acid (TCA) cycle intermediates, amino acids, and fatty acids. Further targeted fatty acid methyl ester (FAME) GC-MS-based profiling of the cellular total fatty acid pools revealed that QS leads to changes associated with decreased membrane fluidity and higher chemical stability. However, not all of the changes we observed were necessarily a direct consequence of QS; liquid chromatography (LC)-MS analyses revealed that polyamine levels were elevated in thelasI rhlImutant, perhaps a response to the absence of QS-dependent adaptations. Our data suggest that QS leads to a global readjustment in central metabolism and provide new insight into the metabolic changes associated with QS during stationary-phase adaptation.IMPORTANCEQuorum sensing (QS) is a transcriptional regulatory mechanism that allows bacteria to coordinate their gene expression profile with the population cell density. The opportunistic human pathogenPseudomonas aeruginosauses QS to control the production of secreted virulence factors. In this study, we show that QS elicits a global “metabolic rewiring” inP. aeruginosa. This metabolic rerouting of fluxes is consistent with a variety of drivers, ranging from altered QS-dependent transcription of “metabolic genes” through to the effect(s) of global “metabolic readjustment” as a consequence of QS-dependent exoproduct synthesis, as well as a general stress response, among others. To our knowledge, this is the first study of its kind to assess the global impact of QS on the metabolome.

scholarly journals Dual Roles of Pseudomonas aeruginosa AlgE in Secretion of the Virulence Factor Alginate and Formation of the Secretion Complex

2013 ◽  
Vol 79 (6) ◽  
pp. 2002-2011 ◽  
Author(s):  
Zahid U. Rehman ◽  
Bernd H. A. Rehm
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Amino Acid ◽  
Cell Envelope ◽  
Extracellular Polysaccharide ◽  
Insertion Mutagenesis ◽  
Amino Acid Residues ◽  
Multiprotein Complex ◽  
Flag Epitope ◽  
Content Type ◽  
Alginate Production

ABSTRACTAlgE is a monomeric 18-stranded β-barrel protein required for secretion of the extracellular polysaccharide alginate inPseudomonas aeruginosa. To assess the molecular mechanism of alginate secretion, AlgE was subjected to site-specific and FLAG epitope insertion mutagenesis. Except for β-strands 6 and 10, epitope insertions into the transmembrane β-strands abolished localization of AlgE to the outer membrane. Interestingly, an epitope insertion into β-strand 10 produced alginate and was only detectable in outer membranes isolated from cells grown on solid media. The deletion of nine C-terminal amino acid residues destabilized AlgE. Replacement of amino acids that constitute the highly electropositive pore constriction showed that individual amino acid residues have a specific function in alginate secretion. Two of the triple mutants (K47E+R353A+R459E and R74E+R362A+R459E) severely reduced alginate production. Mutual stability analysis using thealgEdeletion mutant PDO300ΔalgE(miniCTX) showed the periplasmic alginate biosynthesis proteins AlgK and AlgX were completely destabilized, while the copy number of the inner membrane c-di-GMP receptor Alg44 was reduced. Chromosomal integration ofalgErestored AlgK, AlgX, and Alg44, providing evidence for a multiprotein complex that spans the cell envelope. Periplasmic turn 4 of AlgE was identified as an important region for maintaining the stability of the putative multiprotein complex.

scholarly journals Antimicrobial Activity of, and Cellular Pathways Targeted by, p-Anisaldehyde and Epigallocatechin Gallate in the Opportunistic Human Pathogen Pseudomonas aeruginosa

2019 ◽  
Vol 86 (4) ◽  
Author(s):  
Yetunde Adewunmi ◽  
Sanchirmaa Namjilsuren ◽  
William D. Walker ◽  
Dahlia N. Amato ◽  
Douglas V. Amato ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Antimicrobial Activity ◽  
Essential Oils ◽  
Epigallocatechin Gallate ◽  
Human Pathogen ◽  
Plant Metabolites ◽  
Content Type ◽  
Star Anise ◽  
Cellular Pathways ◽  
Opportunistic Human Pathogen

ABSTRACT Plant-derived aldehydes are constituents of essential oils that possess broad-spectrum antimicrobial activity and kill microorganisms without promoting resistance. In our previous study, we incorporated p-anisaldehyde from star anise into a polymer network called proantimicrobial networks via degradable acetals (PANDAs) and used it as a novel drug delivery platform. PANDAs released p-anisaldehyde upon a change in pH and humidity and controlled the growth of the multidrug-resistant pathogen Pseudomonas aeruginosa PAO1. In this study, we identified the cellular pathways targeted by p-anisaldehyde by generating 10,000 transposon mutants of PAO1 and screened them for hypersensitivity to p-anisaldehyde. To improve the antimicrobial efficacy of p-anisaldehyde, we combined it with epigallocatechin gallate (EGCG), a polyphenol from green tea, and demonstrated that it acts synergistically with p-anisaldehyde in killing P. aeruginosa. We then used transcriptome sequencing to profile the responses of P. aeruginosa to p-anisaldehyde, EGCG, and their combination. The exposure to p-anisaldehyde altered the expression of genes involved in modification of the cell envelope, membrane transport, drug efflux, energy metabolism, molybdenum cofactor biosynthesis, and the stress response. We also demonstrate that the addition of EGCG reversed many p-anisaldehyde-coping effects and induced oxidative stress. Our results provide insight into the antimicrobial activity of p-anisaldehyde and its interactions with EGCG and may aid in the rational identification of new synergistically acting combinations of plant metabolites. Our study also confirms the utility of the thiol-ene polymer platform for the sustained and effective delivery of hydrophobic and volatile antimicrobial compounds. IMPORTANCE Essential oils (EOs) are plant-derived products that have long been exploited for their antimicrobial activities in medicine, agriculture, and food preservation. EOs represent a promising alternative to conventional antibiotics due to their broad-range antimicrobial activity, low toxicity to human commensal bacteria, and capacity to kill microorganisms without promoting resistance. Despite the progress in the understanding of the biological activity of EOs, our understanding of many aspects of their mode of action remains inconclusive. The overarching aim of this work was to address these gaps by studying the molecular interactions between an antimicrobial plant aldehyde and the opportunistic human pathogen Pseudomonas aeruginosa. The results of this study identify the microbial genes and associated pathways involved in the response to antimicrobial phytoaldehydes and provide insights into the molecular mechanisms governing the synergistic effects of individual constituents within essential oils.

scholarly journals Pseudomonas aeruginosa Condensins Support Opposite Differentiation States

2016 ◽  
Vol 198 (21) ◽  
pp. 2936-2944 ◽  
Author(s):  
Hang Zhao ◽  
April L. Clevenger ◽  
Jerry W. Ritchey ◽  
Helen I. Zgurskaya ◽  
Valentin V. Rybenkov
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Biofilm Formation ◽  
Competitive Growth ◽  
Human Pathogen ◽  
Functional Protein ◽  
Content Type ◽  
Bacterial Physiology ◽  
Growth Defects ◽  
Bacterial Pathogenicity ◽  
Opportunistic Human Pathogen

ABSTRACTCondensins play a key role in global chromosome packing.Pseudomonas aeruginosaencodes two condensins, SMC-ScpAB and MksBEF. We report here that the two proteins are involved in the differentiation of the bacterium and impose opposite physiological states. The inactivation of SMC induced a state characterized by increased adhesion to surfaces as well as defects in competitive growth and colony formation. In contrast, MksB-deficient cells were impaired in biofilm formation with no obvious defects during planktonic growth. The phenotype of the double mutant was dominated by the absence of MksB, indicating that the observed growth defects are regulatory in their nature rather than structural. ATPase mutations recapitulated many of the phenotypes of the condensins, indicating their requirement for a functional protein. Additionally, inactivation of condensins dramatically reduced the virulence of the bacterium in a murine model of lung infection. These data demonstrate that condensins are involved in the differentiation ofP. aeruginosaand reveal their importance for pathogenicity.IMPORTANCEAdaptation and differentiation play key roles in bacterial pathogenicity. InPseudomonas aeruginosa, an opportunistic human pathogen, these processes are mediated by the activity of an intricate regulatory network. We describe here novel members of this network, condensins. We show that the twoP. aeruginosacondensins specialize in the establishment of the sessile and planktonic states of the bacterium. Whereas condensins have well-established roles in global chromosome organization, their roles in regulating bacterial physiology have remained unknown. Our data indicate that the two programs may be linked. We further show that condensins are essential for the pathogenicity ofP. aeruginosa.

scholarly journals Complete Genome Sequence of Stenotrophomonas Phage Mendera

2020 ◽  
Vol 9 (1) ◽  
Author(s):  
Kameron D. Garza ◽  
Heather Newkirk ◽  
Russell Moreland ◽  
Carlos F. Gonzalez ◽  
Mei Liu ◽  
...  
Keyword(s):  
Genome Sequence ◽  
Complete Genome Sequence ◽  
Complete Genome ◽  
Stenotrophomonas Maltophilia ◽  
Human Pathogen ◽  
Base Pairs ◽  
Content Type ◽  
Genomic Annotation ◽  
Opportunistic Human Pathogen

Stenotrophomonas maltophilia is an emerging opportunistic human pathogen. In this report, we describe the isolation and genomic annotation of the S. maltophilia-infecting bacteriophage Mendera. A myophage of 159,961 base pairs, Mendera is T4-like and related most closely to Stenotrophomonas phage IME-SM1.

scholarly journals Bacteriophages LIMElight and LIMEzero of Pantoea agglomerans, Belonging to the “phiKMV-Like Viruses”

2011 ◽  
Vol 77 (10) ◽  
pp. 3443-3450 ◽  
Author(s):  
Evelien M. Adriaenssens ◽  
Pieter-Jan Ceyssens ◽  
Vincent Dunon ◽  
Hans-Wolfgang Ackermann ◽  
Johan Van Vaerenbergh ◽  
...  
Keyword(s):  
Soil Samples ◽  
Pantoea Agglomerans ◽  
Open Reading Frames ◽  
Human Pathogen ◽  
Content Type ◽  
Host Diversity ◽  
Double Stranded Dna ◽  
Opportunistic Human Pathogen ◽  
Reading Frames

ABSTRACTPantoea agglomeransis a common soil bacterium used in the biocontrol of fungi and bacteria but is also an opportunistic human pathogen. It has been described extensively in this context, but knowledge of bacteriophages infecting this species is limited. Bacteriophages LIMEzero and LIMElight ofP. agglomeransare lytic phages, isolated from soil samples, belonging to thePodoviridaeand are the firstPantoeaphages of this family to be described. The double-stranded DNA (dsDNA) genomes (43,032 bp and 44,546 bp, respectively) encode 57 and 55 open reading frames (ORFs). Based on the presence of an RNA polymerase in their genomes and their overall genome architecture, these phages should be classified in the subfamily of theAutographivirinae, within the genus of the “phiKMV-like viruses.” Phylogenetic analysis of all the sequenced members of theAutographivirinaesupports the classification of phages LIMElight and LIMEzero as members of the “phiKMV-like viruses” and corroborates the subdivision into the different genera. These data expand the knowledge ofPantoeaphages and illustrate the wide host diversity of phages within the “phiKMV-like viruses.”

scholarly journals Global transcriptome analysis of Stenotrophomonas maltophilia in response to growth at human body temperature

2021 ◽  
Vol 7 (7) ◽  
Author(s):  
Prashant P. Patil ◽  
Sanjeet Kumar ◽  
Amandeep Kaur ◽  
Samriti Midha ◽  
Kanika Bansal ◽  
...  
Keyword(s):  
Body Temperature ◽  
Human Body ◽  
Transcriptome Analysis ◽  
Stenotrophomonas Maltophilia ◽  
Type Species ◽  
Human Pathogen ◽  
Content Type ◽  
Link Type ◽  
Human Body Temperature ◽  
Opportunistic Human Pathogen

Stenotrophomonas maltophilia is a typical example of an environmental originated opportunistic human pathogen, which can thrive at different habitats including the human body and can cause a wide range of infections. It must cope with heat stress during transition from the environment to the human body as the physiological temperature of the human body (37 °C) is higher than environmental niches (22–30 °C). Interestingly, S. rhizophila a phylogenetic neighbour of S. maltophilia within genus Stenotrophomonas is unable to grow at 37 °C. Thus, it is crucial to understand how S. maltophilia is adapted to human body temperature, which could suggest its evolution as an opportunistic human pathogen. In this study, we have performed comparative transcriptome analysis of S. maltophilia grown at 28 and 37 °C as temperature representative for environmental niches and the human body, respectively. RNA-Seq analysis revealed several interesting findings showing alterations in gene-expression levels at 28 and 37 °C, which can play an important role during infection. We have observed downregulation of genes involved in cellular motility, energy production and metabolism, replication and repair whereas upregulation of VirB/D4 type IV secretion system, aerotaxis, cation diffusion facilitator family transporter and LacI family transcriptional regulators at 37 °C. Microscopy and plate assays corroborated altered expression of genes involved in motility. The results obtained enhance our understanding of the strategies employed by S. maltophilia during adaptation towards the human body.

scholarly journals Amino Acid-Mediated Induction of the Basic Amino Acid-Specific Outer Membrane Porin OprD from Pseudomonas aeruginosa

1999 ◽  
Vol 181 (17) ◽  
pp. 5426-5432 ◽  
Author(s):  
Martina M. Ochs ◽  
Chung-Dar Lu ◽  
Robert E. W. Hancock ◽  
Ahmed T. Abdelal
Keyword(s):  
Amino Acids ◽  
Pseudomonas Aeruginosa ◽  
Amino Acid ◽  
Regulatory Protein ◽  
Basic Amino Acid ◽  
Sole Source ◽  
Activation Mechanism ◽  
Beta Lactam ◽  
Mobility Shift ◽  
Carbon And Nitrogen

ABSTRACT Pseudomonas aeruginosa can utilize arginine and other amino acids as both carbon and nitrogen sources. Earlier studies have shown that the specific porin OprD facilitates the diffusion of basic amino acids as well as the structurally analogous beta-lactam antibiotic imipenem. The studies reported here showed that the expression of OprD was strongly induced when arginine, histidine, glutamate, or alanine served as the sole source of carbon. The addition of succinate exerted a negative effect on induction ofoprD, likely due to catabolite repression. The arginine-mediated induction was dependent on the regulatory protein ArgR, and binding of purified ArgR to its operator upstream of theoprD gene was demonstrated by gel mobility shift and DNase assays. The expression of OprD induced by glutamate as the carbon source, however, was independent of ArgR, indicating the presence of more than a single activation mechanism. In addition, it was observed that the levels of OprD responded strongly to glutamate and alanine as the sole sources of nitrogen. Thus, that the expression ofoprD is linked to both carbon and nitrogen metabolism ofPseudomonas aeruginosa.

scholarly journals Efficient Synthesis of Rare Disaccharides by Engineered β-Glucosidase Based on Hydropathy Index

2020 ◽  
Author(s):  
Kangle Niu ◽  
Zhengyao Liu ◽  
Yuhui Feng ◽  
Tianlong Gao ◽  
Zhenzhen Wang ◽  
...  
Keyword(s):  
Amino Acid ◽  
Rational Design ◽  
Large Scale ◽  
Catalytic Site ◽  
Scale Production ◽  
Total Production ◽  
Amino Acid Residues ◽  
Hydropathy Index ◽  
Reverse Hydrolysis ◽  
Hydrolysis Activity

<p>Oligosaccharides have important therapeutic applications. A useful route for oligosaccharides synthesis, especially rare disaccharides, is reverse hydrolysis by <i>β</i>-glucosidase. However, the low conversion efficiency of disaccharides from monosaccharides limits its large-scale production because the equilibrium is biased in the direction of hydrolysis. Based on the analysis of the docking results, we hypothesized that the hydropathy index of key amino acid residues in the catalytic site is closely related with disaccharide synthesis and more hydrophilic residues located in the catalytic site would enhance reverse hydrolysis activity. In this study, positive variants<i> Tr</i>Cel1b<sup>I177S</sup>, <i>Tr</i>Cel1b<sup>I177S/I174S</sup>, and <i>Tr</i>Cel1b<sup>I177S/I174S/W173H</sup>, and one negative variant <i>Tr</i>Cel1b<sup>N240I</sup> were designed according to the <u>H</u>ydropathy <u>I</u>ndex <u>F</u>or <u>E</u>nzyme <u>A</u>ctivity (HIFEA) strategy. The reverse hydrolysis with <i>Tr</i>Cel1b<sup>I177S/I174S/W173H </sup>was accelerated and then the maximum total production (<a>195.8 mg/ml/mg enzyme</a>) of the synthesized disaccharides was increased 3.5-fold compared to that of wildtype. On the contrary, <a><i>Tr</i>Cel1b</a><sup>N240I</sup> lost reverse hydrolysis activity. The results demonstrate that<a> </a><a>the average hydropathy index</a> of <a>the key amino acid residues </a>in the catalytic site of<i> Tr</i>Cel1b is an important factor for the synthesis of laminaribiose, sophorose, and cellobiose. The HIFEA strategy provides a new perspective for the rational design of <i>β</i>-glucosidases used for the synthesis of oligosaccharides.</p>

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