The small regulatory RNA FasX enhances group A Streptococcus virulence and inhibits pilus expression via serotype‐specific targets

Bacterial pathogens commonly show intra‐species variation in virulence factor expression and often this correlates with pathogenic potential. The group A Streptococcus (GAS) produces a small regulatory RNA (sRNA), FasX, which regulates the expression of pili and the thrombolytic agent streptokinase. As GAS serotypes are polymorphic regarding (a) FasX abundance, (b) the fibronectin, collagen, T‐antigen (FCT) region of the genome, which contains the pilus genes (nine different FCT‐types), and (c) the streptokinase‐encoding gene (ska) sequence (two different alleles), we sought to test whether FasX regulates pilus and streptokinase expression in a serotype‐specific manner. Parental, fasX mutant and complemented derivatives of serotype M1 (ska‐2, FCT‐2), M2 (ska‐1, FCT‐6), M6 (ska‐2, FCT‐1) and M28 (ska‐1, FCT‐4) isolates were compared. While FasX reduced pilus expression in each serotype, the molecular basis differed, as FasX bound, and inhibited the translation of, different FCT‐region mRNAs. FasX enhanced streptokinase expression in each serotype, although the degree of regulation varied. Finally, we established that the regulation afforded by FasX enhances GAS virulence, assessed by a model of bacteremia using human plasminogen‐expressing mice. Our data are the first to identify and characterize serotype‐specific regulation by an sRNA in GAS, and to show an sRNA directly contributes to GAS virulence.     

A Key Role for the Urokinase Plasminogen Activator (uPA) in Invasive Group A Streptococcal Infection

Recruitment of the serine protease plasmin is central to the pathogenesis of many bacterial species, including Group A streptococcus (GAS), a leading cause of morbidity and mortality globally. A key process in invasive GAS disease is the ability to accumulate plasmin at the cell surface, however the role of host activators of plasminogen in this process is poorly understood. Here, we demonstrate for the first time that the urokinase-type plasminogen activator (uPA) contributes to plasmin recruitment and subsequent invasive disease initiation in vivo. In the absence of a source of host plasminogen activators, streptokinase (Ska) was required to facilitate cell surface plasmin acquisition by GAS. However, in the absence of Ska, host activators were sufficient to promote cell surface plasmin acquisition by GAS strain 5448 during incubation with plasminogen or human plasma. Furthermore, GAS were able mediate a significant increase in the activation of zymogen pro-uPA in human plasma. In order to assess the contribution of uPA to invasive GAS disease, a previously undescribed transgenic mouse model of infection was employed. Both C57/black 6J, and AlbPLG1 mice expressing the human plasminogen transgene, were significantly more susceptible to invasive GAS disease than uPA−/− mice. The observed decrease in virulence in uPA−/−mice was found to correlate directly with a decrease in bacterial dissemination and reduced cell surface plasmin accumulation by GAS. These findings have significant implications for our understanding of GAS pathogenesis, and research aimed at therapeutic targeting of plasminogen activation in invasive bacterial infections.     

Characterization of Streptokinases from Group A Streptococci Reveals a Strong Functional Relationship That Supports the Coinheritance of Plasminogen-binding M Protein and Cluster 2b Streptokinase

Group A streptococcus (GAS) strains secrete the protein streptokinase (SK), which functions by activating host human plasminogen (hPg) to plasmin (hPm), thus providing a proteolytic framework for invasive GAS strains. The types of SK secreted by GAS have been grouped into two clusters (SK1 and SK2) and one subcluster (SK2a and SK2b). SKs from cluster 1 (SK1) and cluster 2b (SK2b) display significant evolutionary and functional differences, and attempts to relate these properties to GAS skin or pharynx tropism and invasiveness are of great interest. In this study, using four purified SKs from each cluster, new relationships between plasminogen-binding group A streptococcal M (PAM) protein and SK2b have been revealed. All SK1 proteins efficiently activated hPg, whereas all subclass SK2b proteins only weakly activated hPg in the absence of PAM. Surface plasmon resonance studies revealed that the lower affinity of SK2b to hPg served as the basis for the attenuated activation of hPg by SK2b. Binding of hPg to either human fibrinogen (hFg) or PAM greatly enhanced activation of hPg by SK2b but minimally influenced the already effective activation of hPg by SK1. Activation of hPg in the presence of GAS cells containing PAM demonstrated that PAM is the only factor on the surface of SK2b-expressing cells that enabled the direct activation of hPg by SK2b. As the binding of hPg to PAM is necessary for hPg activation by SK2b, this dependence explains the coinherant relationship between PAM and SK2b and the ability of these particular strains to generate the proteolytic activity that disrupts the innate barriers that limit invasiveness.     

Natural selection and evolution of streptococcal virulence genes involved in tissue-specific adaptations

The molecular mechanisms underlying niche adaptation in bacteria are not fully understood. Primary infection by the pathogen group A streptococcus (GAS) takes place at either the throat or the skin of its human host, and GAS strains differ in tissue site preference. Many skin-tropic strains bind host plasminogen via the plasminogen-binding group A streptococcal M protein (PAM) present on the cell surface; inactivation of genes encoding either PAM or streptokinase (a plasminogen activator) leads to loss of virulence at the skin. Unlike PAM, which is present in only a subset of GAS strains, the gene encoding streptokinase (ska) is present in all GAS isolates. In this study, the evolution of the virulence genes known to be involved in skin infection was examined. Most genetic diversity within ska genes was localized to a region encoding the plasminogen-docking domain (beta-domain). The gene encoding PAM displayed strong linkage disequilibrium (P < 0.01) with a distinct phylogenetic cluster of the ska beta-domain-encoding region. Yet, ska alleles of distant taxa showed a history of intragenic recombination, and high intrinsic levels of recombination were found among GAS strains having different tissue tropisms. The data suggest that tissue-specific adaptations arise from epistatic coselection of bacterial virulence genes. Additional analysis of ska genes showed that approximately 4% of the codons underwent strong diversifying selection. Horizontal acquisition of one ska lineage from a commensal Streptococcus donor species was also evident. Together, the data suggest that new phenotypes can be acquired through interspecies recombination between orthologous genes, while constrained functions can be preserved; in this way, orthologous genes may provide a rich and ready source for new phenotypes and thereby play a facilitating role in the emergence of new niche adaptations in bacteria.     

Combined computational and biochemical study reveals the importance of electrostatic interactions between the “pH sensor” and the cation binding site of the sodium/proton antiporter NhaA of Escherichia coli

Sodium proton antiporters are essential enzymes that catalyze the exchange of sodium ions for protons across biological membranes. The crystal structure of NhaA has provided a basis to explore the mechanism of ion exchange and its unique regulation by pH. Here, the mechanism of the pH activation of the antiporter is investigated through functional and computational studies of several variants with mutations in the ion‐binding site (D163, D164). The most significant difference found computationally between the wild type antiporter and the active site variants, D163E and D164N, are low pK_a values of Glu78 making them insensitive to pH. Although in the variant D163N the pK_a of Glu78 is comparable to the physiological one, this variant cannot demonstrate the long‐range electrostatic effect of Glu78 on the pH‐dependent structural reorganization of trans‐membrane helix X and, hence, is proposed to be inactive. In marked contrast, variant D164E remains sensitive to pH and can be activated by alkaline pH shift. Remarkably, as expected computationally and discovered here biochemically, D164E is viable and active in Na⁺/H⁺ exchange albeit with increased apparent K_M. Our results unravel the unique electrostatic network of NhaA that connect the coupled clusters of the “pH sensor” with the binding site, which is crucial for pH activation of NhaA. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Regulatory gene mutation: a driving force behind group a Streptococcus strain‐ and serotype‐specific variation

Data from multiple bacterial pathogens are consistent with regulator‐encoding genes having higher mutation frequencies than the genome average. Such mutations drive both strain‐ and type‐ (e.g., serotype, haplotype) specific phenotypic heterogeneity, and may challenge public health due to the potential of variants to circumvent established treatment and/or preventative regimes. Here, using the human bacterial pathogen the group A Streptococcus (GAS; S. pyogenes) as a model organism, we review the types and regulatory‐, phenotypic‐, and disease‐specific consequences of naturally occurring regulatory gene mutations. Strain‐specific regulator mutations that will be discussed include examples that transform isolates into hyper‐invasive forms by enhancing expression of immunomodulatory virulence factors, and examples that promote asymptomatic carriage of the organism. The discussion of serotype‐specific regulator mutations focuses on serotype M3 GAS isolates, and how the identified rewiring of regulatory networks in this serotype may be contributing to a decades old epidemiological association of M3 isolates with particularly severe invasive infections. We conclude that mutation plays an outsized role in GAS pathogenesis and has clinical relevance. Given the phenotypic variability associated with regulatory gene mutations, the rapid examination of these genes in infecting isolates may inform with respect to potential patient complications and treatment options.     

The β-domain of cluster 2b streptokinase is a major determinant for the regulation of its plasminogen activation activity by cellular plasminogen receptors

Cluster 2b streptokinase (SK2b), secreted by invasive skin-trophic strains of Streptococcus pyogenes (GAS), is a human plasminogen (hPg) activator that optimally functions when human plasma hPg is bound, via its kringle-2 domain, to cognizant bacterial cells through the a1a2 domain of the major cellular hPg receptor, Plasminogen-binding group A streptococcal M-like protein (PAM). Another class of streptokinases (SK1), secreted primarily by GAS strains that possess affinity for pharyngeal infections, does not require PAM-bound hPg for optimal activity. We find herein that replacement of the central β-domain of SK2b with the same module from SK1 reduces the dependency of SK2b on PAM, and the converse is true when the β-domain of SK1 is replaced with this same region of SK2b. These data suggest that simple evolutionary shuttling of protein domains in GAS can be employed by GAS to rapidly generate strains that differ in tissue tropism and invasive capability and allow the bacteria to survive different challenges by the host.     

Numerical taxonomy of psychrotrophic lactic acid bacteria from prepacked meat and meat products

Ninety-four strains of lactic acid bateria isolated from refrigerated, prepacked meat and meat products were together with 59 reference strains of Brochothrix, Lactobacillus, Leuconostoc, Pediococcus and Streptococcus phenotypically classfied, using 96 unit characters. Data were examined using Simple Matching (S_SM) or Jaccard coefficient (S_J), and unweighted pair group algorithm with arithmetic averages. Twenty-three clusters with two or more members were defined at the 84% S_SM-similarity level which corresponded to the S_J-similarity level of 61%. Based on S_SM, most field strains were included in nine clusters, and with three unsignificant exceptions these contained no reference strains. The field clusters were designated Carnobacterium piscicola (cluster 1; 5% of field isolates), Carnobacterium divergens(cluster 2; 9% of field isolates), Leuconostoc (cluster 9; 18% of field isolates) and Lactobacillus (cluster 4, 10, 11, 12, 13 and 14; together 60% of field isolates). The Lactobacillus clusters had many features in common with cluster II of Shaw & Harding (1984). Phenotypical characteristics of major clusters are given. The S_SM and S_J based classifications basically coincided for the field strains; the exception was cluster 4 which now were split in two parts. Fourteen clusters were made up of mainly reference strains (S_SM). Most of them included more than one type strain on species level; exceptions were Brochothrix thermosphacta (cluster 3), Lactobacillus salivarius (cluster 17) and Leuconostoc mesenteroides (cluster 18). Several rearrangements were seen amongst the clusters of the reference strains when S_J, instead of S_SM, was used for clustering.     

Ozone treatment affects pigment precursor metabolism in pine seedlings

Five‐week‐old seedlings of Pinus halepensis Mill. and Pinus brutia Ten. were exposed to air polluted with ozone (O₃) (250 nl l^?1, 12 h day^?1 for 4 days) or to ambient air containing ca 10–20 nl l^?1 O₃, in the light (180 μmol m^?2 s^?1 photosynthetic photon flux density [PPFD], 12 h day^?1) and then fed for 24 h in the light (100 μmol m^?2 s^?1 PPFD) with various radioactive precursors of chlorophyll (Chl) and carotene biosynthesis: 5‐[4‐¹⁴C]‐aminolevulinic acid (¹⁴C‐ALA), l ‐[¹⁴C(U)]‐glutamic acid (¹⁴C‐Glu), or d ,l ‐[2‐¹⁴C]‐mevalonic acid (¹⁴C‐MVA). Pigments were then extracted from cotyledons and fully expanded needles. Chl a and carotene were separated by thin‐layer chromatography and high‐performance liquid chromatography and their specific activities were determined. ¹⁴C‐ALA and ¹⁴C‐Glu labels were incorporated into Chl a and carotene. Exposure to O₃ did not inhibit incorporation of ¹⁴C‐ALA into Chl a molecules, but hydrolysis of Chl a showed that O₃ inhibited phytol labelling of Chl a. Labelling of carotene was also inhibited by O₃, but not when ¹⁴C‐MVA was used as the label. These data suggest that O₃ treatment inhibits (directly or indirectly) the biosynthesis of isoprenoids from products of ALA and Glu metabolism in the plastid, but not from MVA in the cytosol. This inhibition was more prominent when ¹⁴C‐ALA was used as the label than when ¹⁴C‐Glu was the labelling precursor. A significant increase in pheophorbide a, a tetrapyrrole component of Chl a labelling, and a concomitant decrease in phytol labelling was observed following incubation of O₃‐treated pine seedlings with ¹⁴C‐ALA and ¹⁴C‐Glu. Stronger inhibition of carotene biosynthesis and activation of Chl a tetrapyrrole labelling by ¹⁴C‐ALA (in comparison with ¹⁴C‐Glu) indicated that exposure to O₃ inhibits the conversion of ALA to Glu as the first step in ALA catabolism. These results also suggested a more intensive Glu metabolism (in comparison with ALA) for carotene biosynthesis in the cytosol, as well as cooperation between two pathways of isopentenyl diphosphate biosynthesis.     

The Streptococcus pyogenes orphan protein tyrosine phosphatase,SP‐PTP,possesses dual specificity and essential virulence regulatory functions

Group A Streptococcus (GAS) is a human pathogen that causes high morbidity and mortality. GAS lacks a gene encoding tyrosine kinase but contains one encoding tyrosine phosphatase (SP‐PTP). Thus, GAS is thought to lack tyrosine phosphorylation, and the physiological significance of SP‐PTP is, therefore, questionable. Here, we demonstrate that SP‐PTP possesses dual phosphatase specificity for Tyr‐ and Ser/Thr‐phosphorylated GAS proteins, such as Ser/Thr kinase (SP‐STK) and the SP‐STK‐phosphorylated CovR and WalR proteins. Phenotypic analysis of GAS mutants lacking SP‐PTP revealed that the phosphatase activity per se positively regulates growth, cell division and the ability to adhere to and invade host cells. Furthermore, A549 human lung cells infected with GAS mutants lacking SP‐PTP displayed increased Ser‐/Thr‐/Tyr‐phosphorylation. SP‐PTP also differentially regulates the expression of ～50% of the total GAS genes, including several virulence genes potentially through the two‐component regulators, CovR, WalR and PTS/HPr regulation of Mga. Although these mutants exhibit attenuated virulence, a GAS mutant overexpressing SP‐PTP is hypervirulent. Our study provides the first definitive evidence for the presence and importance of Tyr‐phosphorylation in GAS and the relevance of SP‐PTP as an important therapeutic target.     

The maintenance of high affinity plasminogen binding by group A streptococcal plasminogen-binding M-like protein is mediated by arginine and histidine residues within the a1 and a2 repeat domains

Subversion of the plasminogen activation system is implicated in the virulence of group A streptococci (GAS). GAS displays receptors for the human zymogen plasminogen on the cell surface, one of which is the plasminogen-binding group A streptococcal M-like protein (PAM). The plasminogen binding domain of PAM is highly variable, and this variation has been linked to host selective immune pressure. Site-directed mutagenesis of full-length PAM protein from an invasive GAS isolate was undertaken to assess the contribution of residues in the a1 and a2 repeat domains to plasminogen binding function. Mutagenesis to alanine of key plasminogen binding lysine residues in the a1 and a2 repeats (Lys98 and Lys111) did not abrogate plasminogen binding by PAM nor did additional mutagenesis of Arg101 and His102 and Glu104, which have previously been implicated in plasminogen binding. Plasminogen binding was only abolished with the additional mutagenesis of Arg114 and His115 to alanine. Furthermore, mutagenesis of both arginine (Arg101 and Arg114) and histidine (His102 and His115) residues abolished interaction with plasminogen despite the presence of Lys98 and Lys111 in the binding repeats. This study shows for the first time that residues Arg101, Arg114, His102, and His115 in both the a1 and a2 repeat domains of PAM can mediate high affinity plasminogen binding. These data suggest that highly conserved arginine and histidine residues may compensate for variation elsewhere in the a1 and a2 plasminogen binding repeats, and may explain the maintenance of high affinity plasminogen binding by naturally occurring variants of PAM.     

Case report of clinical salmonellosis by Salmonella Typhimurium that occurred in Portuguese children

Aims: Aim of this study is to characterize clinical isolates of Salmonella Typhimurium that occurred in Portuguese children on the basis of their virulence and antimicrobial resistance profiles and pulsed‐field gel electrophoresis typing and to analyse possible strain relatedness. Methods and Results: Different Salmonella serotypes were isolated from clinical cases of salmonellosis that had occurred in two Portuguese hospitals (a total of 259 isolates). All Salm. Typhimurium strains, with the age of the patients known, (total of 26 isolates) were selected for this study. These isolates were characterized for their virulence gene profiles (agfA, iroB, slyA, hin/H2, spv), antimicrobial resistance profiles and investigated for the occurrence of multidrug‐resistant Salm. Typhimurium DT 104 by PCR. Salmonella isolates showed high rates of resistance to four or more antibiotics, 100% resistance to sulfadiazine and a high percentage of strains with the resistance profile of Salm. Typhimurium DT 104, two of them with this phage type (determined by PCR). A relationship between some clusters and their resistance and virulence profiles was detected, each cluster having the same profile. Conclusions: This study showed high‐antibiotic resistance of the Salmonella strains investigated, and the presence of multidrug‐resistant Salm. Typhimurium DT104 in infections of Portuguese children. Significance and Impact of the Study: Study is based on regarding the increase in antibiotic resistance by Salmonella strains isolated from infections in Portuguese children and on the presence of Salm. Typhimurium DT 104 circulating in Portugal.     

Identification of a New Exosite Involved in Catalytic Turnover by the Streptokinase-Plasmin Activator Complex during Human Plasminogen Activation

With the goal of identifying hitherto unknown surface exosites of streptokinase involved in substrate human plasminogen recognition and catalytic turnover, synthetic peptides encompassing the 170 loop (CQFTPLNPDDDFRPGLKDTKLLC) in the β-domain were tested for selective inhibition of substrate human plasminogen activation by the streptokinase-plasmin activator complex. Although a disulfide-constrained peptide exhibited strong inhibition, a linear peptide with the same sequence, or a disulfide-constrained variant with a single lysine to alanine mutation showed significantly reduced capabilities of inhibition. Alanine-scanning mutagenesis of the 170 loop of the β-domain of streptokinase was then performed to elucidate its importance in streptokinase-mediated plasminogen activation. Some of the 170 loop mutants showed a remarkable decline in k_cat without any alteration in apparent substrate affinity (K_m) as compared with wild-type streptokinase and identified the importance of Lys¹⁸⁰ as well as Pro¹⁷⁷ in the functioning of this loop. Remarkably, these mutants were able to generate amidolytic activity and non-proteolytic activation in “partner” plasminogen as wild-type streptokinase. Moreover, cofactor activities of the 170 loop mutants, pre-complexed with plasmin, against microplasminogen as the substrate showed a similar pattern of decline in k_cat as that observed in the case of full-length plasminogen, with no concomitant change in K_m. These results strongly suggest that the 170 loop of the β-domain of streptokinase is important for catalysis by the streptokinase-plasmin(ogen) activator complex, particularly in catalytic processing/turnover of substrate, although it does not seem to contribute significantly toward enzyme-substrate affinity per se.     

Emergence of carbapenem non‐susceptible multidrug resistant Acinetobacter baumannii strains of clonal complexes 103B and 92B harboring OXA‐type carbapenemases and metallo‐β‐lactamases in Southern India

The molecular epidemiology and carbapenem resistance mechanisms of clinical isolates of Acinetobacter baumannii obtained from a south Indian tertiary care hospital were investigated by repetitive extragenic palindromic sequence PCR (REP‐PCR) and multi‐locus sequence typing (MLST). Analysis of resistant determinants was achieved by PCR screening for the presence of genes encoding OXA‐carbapenemases, metallo‐β‐lactamases (MBLs) and efflux pumps. REP‐PCR generated around eight clusters of high heterogeneity; of these, two major clusters (I and V) appeared to be clonal in origin. Analysis of representative isolates from different clusters by MLST revealed that most of the isolates belonged to sequence type 103 of CC103^B. Second most prevalent ST belonged to clonal complex (CC) 92^B which is also referred to as international clone II. Most of the isolates were multi‐drug resistant, being susceptible only to polymyxin‐B and newer quinolones. Class D β‐lactamases such as bla_{OXA‐51‐like} (100%), bla_{OXA‐23‐like} (56.8%) and bla_{OXA‐24‐like} (14.8%) were found to be predominant, followed by a class B β‐lactamase, namely bla_IMP‐1 (40.7%); none of the isolates had bla_OXA‐58 like, bla_NDM‐1 or bla_SIM‐1. Genes of efflux‐pump adeABC were predominant, most of isolates being biofilm producers that were PCR‐positive for autoinducer synthase gene (>94%). Carbapenem non‐susceptible isolates were highly diverse and present throughout the hospital irrespective of type of ward or intensive care unit. Although previous reports have documented diverse resistant mechanisms in A. baumannii, production of MBL and OXA‐type of carbapenamases were found to be the predominant mechanism(s) of carbapenem resistance identified in strains isolated from Southern India.     

Characterization of Phytophthora cinnamomi populations from ornamental plants in South Carolina,USA

Abstract

Fifty-one isolates of Phytophthora cinnamomi isolated from ornamental plants in South Carolina, USA, between 1995 and 2000 were characterized by sporangium morphology, mating type, sensitivity to the fungicide mefenoxam, fatty acid methyl ester (FAME) profile analysis, and amplified fragment length polymorphism (AFLP) analysis. Sporangium shapes were predominantly ovoid to ellipsoid, and size averaged 65.5×40.3 μm (length×breadth) with average length/breadth ratio of 1.6. Forty-nine isolates were the A2 mating type with only two A1 isolates found. This is the first report of the A1 mating type of P. cinnamomi in South Carolina. All isolates were sensitive to mefenoxam and EC₅₀ values for all isolates were less than 0.2 μg ml^?1. FAMEs of each isolate were analysed by gas chromatography and revealed five major fatty acids: myristic (14:0), palmitic (16:0), linoleic (18:2ω6c), oleic (18:1ω9c), and eicosapentaenoic (20:5ω3c) acids. These five fatty acids accounted for more than 80% of FAME profiles. Cluster analysis of FAME profiles showed that individual isolates had unique pattern that could be divided into four major clusters. AFLP analysis based on 200 informative loci also separated isolates into four major clusters. A1 isolates were different from all A2 isolates. The percentage of polymorphic loci (10.5%) and Nei's gene diversity (0.0435) were much higher for the two A1 isolates than for any cluster of A2 isolates even though A2 isolates had more isolates within a cluster. A2 isolates exhibited relatively little genetic variation overall, which suggests that these isolates may have come from a common source.     

Characterizing the role of tissue-type plasminogen activator in a mouse model of Group A streptococcal infection

Plasmin(ogen) acquisition is critical for invasive disease initiation by Streptococcus pyogenes (GAS). Host urokinase plasminogen activator (uPA) plays a role in mediating plasminogen activation for GAS dissemination, however the contribution of tissue-type plasminogen activator (tPA) to GAS virulence is unknown. Using novel tPA-deficient ALBPLG1 mice, our study revealed no difference in mouse survival, bacterial dissemination or the pathology of GAS infection in the absence of tPA in AlbPLG1/tPA^?/? mice compared to AlbPLG1 mice. This study suggests that tPA has a limited role in this humanized model of GAS infection, further highlighting the importance of its counterpart uPA in GAS disease.     

Directed evolution of Aspergillus niger glucoamylase to increase thermostability

Using directed evolution and site‐directed mutagenesis, we have isolated a highly thermostable variant of Aspergillus niger glucoamylase (GA), designated CR2‐1 . CR2‐1 includes the previously described mutations Asn20Cys and Ala27Cys (forming a new disulfide bond), Ser30Pro, Thr62Ala, Ser119Pro, Gly137Ala, Thr290Ala, His391Tyr and Ser436Pro. In addition, CR2‐1 includes several new putative thermostable mutations, Val59Ala, Val88Ile, Ser211Pro, Asp293Ala, Thr390Ser, Tyr402Phe and Glu408Lys, identified by directed evolution. CR2‐1 GA has a catalytic efficiency (k_cat/K_m) at 35°C and a specific activity at 50°C similar to that of wild‐type GA. Irreversible inactivation tests indicated that CR2‐1 increases the free energy of thermoinactivation at 80°C by 10 kJ mol^?1 compared with that of wild‐type GA. Thus, CR2‐1 is more thermostable (by 5 kJ mol^?1 at 80°C) than the most thermostable A. niger GA variant previously described, THS8 . In addition, Val59Ala and Glu408Lys were shown to individually increase the thermostability in GA variants by 1 and 2 kJ mol^?1, respectively, at 80°C.