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Background: Cellobiose dehydrogenase (CDH) is an extracellular fungal oxidoreductase with multiple functions in plant biomass degradation. Its primary function as an auxiliary enzyme of lytic polysaccharide monooxygenase (LPMO) facilitates the efficient depolymerization of cellulose, hemicelluloses and other carbohydrate-based polymers. The synergistic action of CDH and LPMO that supports biomass-degrading hydrolases holds significant promise to harness renewable resources for the production of biofuels, chemicals, and modified materials in an environmentally sustainable manner. While previous phylogenetic analyses have identified four distinct classes of CDHs, only class I and II have been biochemically characterized so far., Results: Following a comprehensive database search aimed at identifying CDH sequences belonging to the so far uncharacterized class III for subsequent expression and biochemical characterization, we have curated an extensive compilation of putative CDH amino acid sequences. A sequence similarity network analysis was used to cluster them into the four distinct CDH classes. A total of 1237 sequences encoding putative class III CDHs were extracted from the network and used for phylogenetic analyses. The obtained phylogenetic tree was used to guide the selection of 11 cdhIII genes for recombinant expression in Komagataella phaffii. A small-scale expression screening procedure identified a promising cdhIII gene originating from the plant pathogen Fusarium solani (FsCDH), which was selected for expression optimization by signal peptide shuffling and subsequent production in a 5-L bioreactor. The purified FsCDH exhibits a UV-Vis spectrum and enzymatic activity similar to other characterized CDH classes., Conclusion: The successful production and functional characterization of FsCDH proved that class III CDHs are catalytical active enzymes resembling the key properties of class I and class II CDHs. A detailed biochemical characterization based on the established expression and purification strategy can provide new insights into the evolutionary process shaping CDHs and leading to their differentiation into the four distinct classes. The findings have the potential to broaden our understanding of the biocatalytic application of CDH and LPMO for the oxidative depolymerization of polysaccharides., (© 2024. The Author(s).)

Biological conversion of plant biomass depends on peroxygenases and peroxidases acting on insoluble polysaccharides and lignin. Among these are cellulose- and hemicellulose-degrading lytic polysaccharide monooxygenases (LPMOs), which have revolutionized our concept of biomass degradation. Major obstacles limiting mechanistic and functional understanding of these unique peroxygenases are their complex and insoluble substrates and the hard-to-measure H 2 O 2 consumption, resulting in the lack of suitable kinetic assays. We report a versatile and robust electrochemical method for real-time monitoring and kinetic characterization of LPMOs and other H 2 O 2 -dependent interfacial enzymes based on a rotating disc electrode for the sensitive and selective quantitation of H 2 O 2 at biologically relevant concentrations. The H 2 O 2 sensor works in suspensions of insoluble substrates as well as in homogeneous solutions. Our characterization of multiple LPMOs provides unprecedented insights into the substrate specificity, kinetics, and stability of these enzymes. High turnover and total turnover numbers demonstrate that LPMOs are fast and durable biocatalysts., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

The function of cellobiose dehydrogenase (CDH) in biosensors, biofuel cells, and as a physiological redox partner of lytic polysaccharide monooxygenase (LPMO) is based on its role as an electron donor. Before donating electrons to LPMO or electrodes, an interdomain electron transfer from the catalytic FAD-containing dehydrogenase domain to the electron shuttling cytochrome domain of CDH is required. This study investigates the role of two crucial amino acids located at the dehydrogenase domain on domain interaction and interdomain electron transfer by structure-based engineering. The electron transfer kinetics of wild-type Myriococcum thermophilum CDH and its variants M309A, R698S, and M309A/R698S were analyzed by stopped-flow spectrophotometry and structural effects were studied by small-angle X-ray scattering. The data show that R698 is essential to pull the cytochrome domain close to the dehydrogenase domain and orient the heme propionate group towards the FAD, while M309 is an integral part of the electron transfer pathway - its mutation reducing the interdomain electron transfer 10-fold. Structural models and molecular dynamics simulations pinpoint the action of these two residues on the domain interaction and interdomain electron transfer., (© 2023 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

The interdomain electron transfer (IET) between the catalytic flavodehydrogenase domain and the electron-transferring cytochrome domain of cellobiose dehydrogenase (CDH) plays an essential role in biocatalysis, biosensors and biofuel cells, as well as in its natural function as an auxiliary enzyme of lytic polysaccharide monooxygenase. We investigated the mobility of the cytochrome and dehydrogenase domains of CDH, which is hypothesised to limit IET in solution by small angle X-ray scattering (SAXS). CDH from Myriococcum thermophilum (syn. Crassicarpon hotsonii, syn. Thermothelomyces myriococcoides) was probed by SAXS to study the CDH mobility at different pH and in the presence of divalent cations. By comparison of the experimental SAXS data, using pair-distance distribution functions and Kratky plots, we show an increase in CDH mobility at higher pH, indicating alterations of domain mobility. To further visualise CDH movement in solution, we performed SAXS-based multistate modelling. Glycan structures present on CDH partially masked the resulting SAXS shapes, we diminished these effects by deglycosylation and studied the effect of glycoforms by modelling. The modelling shows that with increasing pH, the cytochrome domain adopts a more flexible state with significant separation from the dehydrogenase domain. On the contrary, the presence of calcium ions decreases the mobility of the cytochrome domain. Experimental SAXS data, multistate modelling and previously reported kinetic data show how pH and divalent ions impact the closed state necessary for the IET governed by the movement of the CDH cytochrome domain., (© 2023 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Lytic polysaccharide monooxygenase (LPMO) is a monocopper-dependent enzyme that cleaves glycosidic bonds by using an oxidative mechanism. In nature, they act in concert with cellobiohydrolases to facilitate the efficient degradation of lignocellulosic biomass. After more than a decade of LPMO research, it has become evident that LPMOs are abundant in all domains of life and fulfill a diverse range of biological functions. Independent of their biological function and the preferred polysaccharide substrate, studying and characterizing LPMOs is tedious and so far mostly relied on the discontinuous analysis of the solubilized reaction products by HPLC/MS-based methods. In the absence of appropriate substrates, LPMOs can engage in two off-pathway reactions, i.e., an oxidase and a peroxidase-like activity. These futile reactions have been exploited to set up easy-to-use continuous spectroscopic assays. As the natural substrates of newly discovered LPMOs are often unknown, widely applicable, simple, reliable, and robust spectroscopic assays are required to monitor LPMO expression and to perform initial biochemical characterizations, e.g., thermal stability measurements. Here we provide detailed descriptions and practical protocols to perform continuous photometric assays using either 2,6-dimethoxyphenol (2,6-DMP) or hydrocoerulignone as colorimetric substrates as a broadly applicable assay for a range of LPMOs. In addition, a turbidimetric measurement is described as the currently only method available to continuously monitor LPMOs acting on amorphous cellulose., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Lytic polysaccharide monooxygenase (LPMO) supports biomass hydrolysis by increasing saccharification efficiency and rate. Recent studies demonstrate that H 2 O 2 rather than O 2 is the cosubstrate of the LPMO-catalyzed depolymerization of polysaccharides. Some studies have questioned the physiological relevance of the H 2 O 2 -based mechanism for plant cell wall degradation. This study reports the localized and time-resolved determination of LPMO activity on poplar wood cell walls by measuring the H 2 O 2 concentration in their vicinity with a piezo-controlled H 2 O 2 microsensor. The investigated Neurospora crassa LPMO binds to the inner cell wall layer and consumes enzymatically generated H 2 O 2 . The results point towards a high catalytic efficiency of LPMO at a low H 2 O 2 concentration that auxiliary oxidoreductases in fungal secretomes can easily generate. Measurements with a glucose microbiosensor additionally demonstrate that LPMO promotes cellobiohydrolase activity on wood cell walls and plays a synergistic role in the fungal extracellular catabolism and in industrial biomass degradation., (© 2022. The Author(s).)

Lignocelluloytic enzymes are industrially applied as biocatalysts for the deconstruction of recalcitrant plant biomass. To study their biocatalytic and physiological function, the assessment of their binding behavior and spatial distribution on lignocellulosic material is a crucial prerequisite. In this study, selected hydrolases and oxidoreductases from the white rot fungus Phanerochaete chrysosporium were localized on model substrates as well as poplar wood by confocal laser scanning microscopy. Two different detection approaches were investigated: direct tagging of the enzymes and tagging specific antibodies generated against the enzymes. Site-directed mutagenesis was employed to introduce a single surface-exposed cysteine residue for the maleimide site-specific conjugation. Specific polyclonal antibodies were produced against the enzymes and were labeled using N-hydroxysuccinimide (NHS) ester as a cross-linker. Both methods allowed the visualization of cell wall-bound enzymes but showed slightly different fluorescent yields. Using native poplar thin sections, we identified the innermost secondary cell wall layer as the preferential attack point for cellulose-degrading enzymes. Alkali pretreatment resulted in a partial delignification and promoted substrate accessibility and enzyme binding. The methods presented in this study are suitable for the visualization of enzymes during catalytic biomass degradation and can be further exploited for interaction studies of lignocellulolytic enzymes in biorefineries.

To efficiently decompose biomass, fungi have developed various enzymatic and non-enzymatic strategies and are a source of versatile biocatalysts. The endoglucanases in glycosyl hydrolase CAZy family 45 (GH45) are known for their small size, a high thermostability and a broad substrate specificity that has been employed in textile and detergent industries. Here we report the heterologous expression and characterisation of an GH45 endoglucanase from the brown rot Fomitopsis pinicola and its direct comparison to an already characterised GH45 from the white rot Phanerochaete chrysosporium. Both enzymes were recombinantly expressed in Pichia pastoris and purified by two chromatographic steps. The biochemical characterisation highlighted the acidophilic character, with an optimal pH of 4, and a preference for amorphous substrates as carboxymethyl cellulose (CMC) and substrates containing β-1,4-glucans rather than the previously reported β-1,3/1,4-glucans lichenan and β-glucan. The dominating products from β-1,4-glucans were C3-C6 oligosaccharides, whereas from β-1,3/1,4-glucans glucose was the main reaction product. From the characterisation no differences between the brown rot and the white rot GH45 was evident., (Copyright © 2022. Published by Elsevier Inc.)

Enzymatic biofuel cells utilize oxidoreductases as highly specific and highly active electrocatalysts to convert a fuel and an oxidant even in complex biological matrices like hydrolysates or physiological fluids into electric energy. The hemoflavoenzyme cellobiose dehydrogenase is investigated as a versatile bioelectrocatalyst for the anode reaction of biofuel cells, because it is robust, converts a range of different carbohydrates, and can transfer electrons to the anode by direct electron transfer or via redox mediators. The versatility of cellobiose dehydrogenase has led to the development of various electrode modifications to create biofuel cells and biosupercapacitors that are capable to power small electronic devices like biosensors and connect them wireless to a receiver., (Copyright © 2021. Published by Elsevier Ltd.)

The first lytic polysaccharide monooxygenase (LPMO) detected in the genome of the widespread ascomycete Talaromyces amestolkiae (TamAA9A) has been successfully expressed in Pichia pastoris and characterized. Molecular modeling of TamAA9A showed a structure similar to those from other AA9 LPMOs. Although fungal LPMOs belonging to the genera Penicillium or Talaromyces have not been analyzed in terms of regioselectivity, phylogenetic analyses suggested C1/C4 oxidation which was confirmed by HPAEC. To ascertain the function of a C-terminal linker-like region present in the wild-type sequence of the LPMO, two variants of the wild-type enzyme, one without this sequence and one with an additional C-terminal carbohydrate binding domain (CBM), were designed. The three enzymes (native, without linker and chimeric variant with a CBM) were purified in two chromatographic steps and were thermostable and active in the presence of H 2 O 2 . The transition midpoint temperature of the wild-type LPMO (Tm = 67.7 °C) and its variant with only the catalytic domain (Tm = 67.6 °C) showed the highest thermostability, whereas the presence of a CBM reduced it (Tm = 57.8 °C) and indicates an adverse effect on the enzyme structure. Besides, the potential of the different T. amestolkiae LPMO variants for their application in the saccharification of cellulosic and lignocellulosic materials was corroborated.

Glyoxal oxidase (GLOX) is an extracellular source of H 2 O 2 in white-rot secretomes, where it acts in concert with peroxidases to degrade lignin. It has been reported that GLOX requires activation prior to catalytic turnover and that a peroxidase system can fulfill this task. In this study, we verify that an oxidation product of horseradish peroxidase, the radical cation of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), is an activator for GLOX. A spectroelectrochemical cell was used to generate the activating radical species, to continuously measure its concentration, and to simultaneously measure the catalytic activity of GLOX based on its O 2 consumption. The results show that GLOX can undergo multiple catalytic turnovers upon activation and that activity increases with the activator concentration. However, we also found that the ABTS cation radical can serve as an electron acceptor which becomes visible in the absence of O 2 . Furthermore, GLOX activity is highly restrained by the naturally occurring, low O 2 concentration. We conclude that GLOX is indeed an auxiliary enzyme for H 2 O 2 production in white-rot secretomes. Its turnover rate is strongly regulated by the availability of O 2 and the radical generating activity of peroxidases present in the secretome, which acts as a feedback loop for GLOX activity., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Laccase is predominantly found in lignin degrading filamentous white rot fungi, where it is involved in the oxidative degradation of this recalcitrant heteropolymer. In brown rot fungi it is much less prevalent: laccases from only a few brown rots have been detected and only two have been characterized. This study tries to understand the role of this ligninolytic enzyme in brown rots by investigating the catalytic properties of laccases secreted by Fomitopsis pinicola FP58527 SS1. When grown on either poplar or spruce wood blocks, several laccases were detected in the secretome. Two of them (FpLcc1 and FpLcc2) were heterologously produced using Trichoderma reesei QM9414 Δxyr1 as expression host and purified to homogeneity by consecutive steps of hydrophobic interaction, anion exchange and size exclusion chromatography. With the substrates 2,2-azino-bis(3-ethylthiazoline-6-sulfonate) (ABTS), 2,6-dimethoxyphenol (2,6-DMP) and guaiacol both laccases showed similar, low pH-optima below 3 for ABTS and 2,6-DMP and at pH 3.5 for guaiacol which is at the acidic end of laccases isolated from white rot fungi. The determined K M values were low while k cat values measured at acidic conditions were comparable to those reported for other laccases from white rot fungi. While both enzymes showed a moderate decrease in activity in the presence of oxalic and citric acid FpLcc2 was activated by acetic acid up to 3.7 times. This activation effect is much more pronounced at pH 5.0 compared to pH 3.0 and could already be observed at a concentration of 1 mM acetic acid., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Enzymatic hydrolysis of lignocellulosic biomass for biofuel production relies on complex multi-enzyme ensembles. Continuous and accurate measurement of the released key products is crucial in optimizing the industrial degradation process and also investigating the activity and interaction between the involved enzymes and the insoluble substrate. Amperometric biosensors have been applied to perform continuous cellobiose measurements during the enzymatic hydrolysis of pure cellulose powders. The oxygen-sensitive mediators used in these biosensors restricted their function under physiological or industrial conditions. Also, the combined measurements of the hydrolysis products cellobiose and glucose require a high selectivity of the biorecognition elements. We employed an [Os(2,2'-bipyridine) 2 Cl]Cl-modified polymer and cellobiose dehydrogenase to fabricate a cellobiose biosensor, which can accurately and specifically detect cellobiose even in the presence of oxygen and the other main product glucose. Additionally, a glucose biosensor was fabricated to simultaneously measure glucose produced from cellobiose by β-glucosidases. The cellobiose and glucose biosensors work at applied potentials of +0.25 and +0.45 V versus Ag|AgCl (3 M KCl), respectively, and can selectively detect their substrate. Both biosensors were used in combination to monitor the hydrolysis of pure cellulose of low crystallinity or industrial corncob samples. The obtained results correlate with the high-performance liquid chromatography pulsed amperometric detection analysis and demonstrate that neither oxygen nor the presence of redox-active compounds from the lignin fraction of the corncob interferes with the measurements.

In the secretome of Phanerochaete chrysosporium, a white-rot fungus serving as a model organism to elucidate lignocellulose deconstruction, the copper containing metalloprotein glyoxal oxidase (GLOX) is potentially involved in the crucial production of hydrogen peroxide to fuel and initiate oxidative biomass degradation by lignin-degrading peroxidases. Its ability to oxidize a variety of aldehydes and α-hydroxy carbonyls with the concomitant reduction of dioxygen to hydrogen peroxide has attracted attention for its application as green biocatalyst in different industrial fields. Here we report and compare two efficient processes for the heterologous production of GLOX from P. chrysosporium using the well-established methanolytic yeast Pichia pastoris and the filamentous fungus Trichoderma reesei as expression hosts with subsequent purification by anion exchange and hydrophobic interaction chromatography. Both processes were shown to be suitable for the production of the target protein at high levels. GLOX produced in T. reesei carries mainly Man5 glycosylation while the enzyme produced in P. pastoris exhibits the typical high-mannose type N-glycosylation. The enzyme expressed in P. pastoris showed slightly higher specific activities which correlates with the higher copper loading of 65.5 % compared to 51.9 % for the protein from T. reesei. The pH optimum for both recombinant proteins was 6.0, however, GLOX activity was found to be highly affected by different buffer species. Both enzymes showed very similar substrate affinities and turnover numbers with the highest catalytic efficiency observed for methylglyoxal. GLOX from both expression hosts is therefore a suitable enzyme for further mechanistic characterization and application studies., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Cellobiose dehydrogenase from Phanerochaete chrysosporium (PcCDH) is a key enzyme in lignocellulose depolymerization, biosensors and biofuel cells. For these applications, it should retain important molecular and catalytic properties when recombinantly expressed. While homologous expression is time-consuming and the prokaryote Escherichia coli is not suitable for expression of the two-domain flavocytochrome, the yeast Pichia pastoris is hyperglycosylating the enzyme. Fungal expression hosts like Aspergillus niger and Trichoderma reesei were successfully used to express CDH from the ascomycete Corynascus thermophilus. This study describes the expression of basidiomycetes PcCDH in T. reesei (PcCDH Tr ) and the detailed comparison of its molecular, catalytic and electrochemical properties in comparison with PcCDH expressed by P. chrysosporium and P. pastoris (PcCDH Pp )., Results: PcCDH Tr was recombinantly produced with a yield of 600 U L -1 after 4 days, which is fast compared to the secretion of the enzyme by P. chrysosporium. PcCDH Tr and PcCDH were purified to homogeneity by two chromatographic steps. Both enzymes were comparatively characterized in terms of molecular and catalytic properties. The pH optima for electron acceptors are identical for PcCDH Tr and PcCDH. The determined FAD cofactor occupancy of 70% for PcCDH Tr is higher than for other recombinantly produced CDHs and its catalytic constants are in good accordance with those of PcCDH. Mass spectrometry showed high mannose-type N-glycans on PcCDH, but only single N-acetyl-D-glucosamine additions at the six potential N-glycosylation sites of PcCDH Tr , which indicates the presence of an endo-N-acetyl-β-D-glucosaminidase in the supernatant., Conclusions: Heterologous production of PcCDH Tr is faster and the yield higher than secretion by P. chrysosporium. It also does not need a cellulose-based medium that impedes efficient production and purification of CDH by binding to the polysaccharide. The obtained high uniformity of PcCDH Tr glycoforms will be very useful to investigate electron transfer characteristics in biosensors and biofuel cells, which are depending on the spatial restrictions inflicted by high-mannose N-glycan trees. The determined catalytic and electrochemical properties of PcCDH Tr are very similar to those of PcCDH and the FAD cofactor occupancy is good, which advocates T. reesei as expression host for engineered PcCDH for biosensors and biofuel cells.

Future biorefineries are facing the challenge to separate and depolymerize biopolymers into their building blocks for the production of biofuels and basic molecules as chemical stock. Fungi have evolved lignocellulolytic enzymes to perform this task specifically and efficiently, but a detailed understanding of their heterogeneous reactions is a prerequisite for the optimization of large-scale enzymatic biomass degradation. Here, we investigate the binding of cellulolytic enzymes onto biopolymers by surface plasmon resonance (SPR) spectroscopy for the fast and precise characterization of enzyme adsorption processes. Using different sensor architectures, SPR probes modified with regenerated cellulose as well as with lignin films were prepared by spin-coating techniques. The modified SPR probes were analyzed by atomic force microscopy and static contact angle measurements to determine physical and surface molecular properties. SPR spectroscopy was used to study the activity and affinity of Trichoderma reesei cellobiohydrolase I (CBHI) glycoforms on the modified SPR probes. N-glycan removal led to no significant change in activity or cellulose binding, while a slightly higher tendency for non-productive binding to SPR probes modified with different lignin fractions was observed. The results suggest that the main role of the N-glycosylation in CBHI is not to prevent non-productive binding to lignin, but probably to increase its stability against proteolytic degradation. The work also demonstrates the suitability of SPR-based techniques for the characterization of the binding of lignocellulolytic enzymes to biomass-derived polymers., Supplementary Information: The online version contains supplementary material available at 10.1007/s10570-021-04002-6., Competing Interests: Conflict of interestThe authors have no conflict of interest to declare that are relevant to the content of this article., (© The Author(s) 2021.)

Cellobiose dehydrogenase (CDH) is an extracellular hemoflavoenzyme secreted by fungi to assist lignocellulolytic enzymes in biomass degradation. Its catalytic flavodehydrogenase (DH) domain is a member of the glucose-methanol-choline oxidoreductase family similar to glucose oxidase. The catalytic domain is linked to an N-terminal electron transferring cytochrome (CYT) domain which interacts with lytic polysaccharide monooxygenase (LPMO) in oxidative cellulose and hemicellulose depolymerization. Based on CDH sequence analysis, four phylogenetic classes were defined. CDHs in these classes exhibit different structural and catalytic properties in regard to cellulose binding, substrate specificity, and the pH optima of their catalytic reaction or the interdomain electron transfer between the DH and CYT domain. The structure, reaction mechanism and kinetics of CDHs from Class-I and Class-II have been characterized in detail and recombinant expression allows the application in many areas, such as biosensors, biofuel cells biomass hydrolysis, biosynthetic processes, and the antimicrobial functionalization of surfaces., (© 2020 Elsevier Inc. All rights reserved.)

In past years, new lytic polysaccharide monooxygenases (LPMOs) have been discovered as distinct in their substrate specificity. Their unconventional, surface-exposed catalytic sites determine their enzymatic activities, while binding sites govern substrate recognition and regioselectivity. An additional factor influencing activity is the presence or absence of a family 1 carbohydrate binding module (CBM1) connected via a linker to the C-terminus of the LPMO. This study investigates the changes in activity induced by shortening the second active site segment (Seg2) or removing the CBM1 from Neurospora crassa LPMO9C. Nc LPMO9C and generated variants have been tested on regenerated amorphous cellulose (RAC), carboxymethyl cellulose (CMC) and xyloglucan (XG) using activity assays, conversion experiments and surface plasmon resonance spectroscopy. The absence of CBM1 reduced the binding affinity and activity of Nc LPMO9C, but did not affect its regioselectivity. The linker was found important for the thermal stability of Nc LPMO9C and the CBM1 is necessary for efficient binding to RAC. Wild-type Nc LPMO9C exhibited the highest activity and strongest substrate binding. Shortening of Seg2 greatly reduced the activity on RAC and CMC and completely abolished the activity on XG. This demonstrates that Seg2 is indispensable for substrate recognition and the formation of productive enzyme-substrate complexes.

Fungal high redox potential laccases are proposed as cathodic biocatalysts in implantable enzymatic fuel cells to generate high cell voltages. Their application is limited mainly through their acidic pH optimum and chloride inhibition. This work investigates evolutionary and engineering strategies to increase the pH optimum of a chloride-tolerant, high redox potential laccase from the ascomycete Botrytis aclada. The laccase was subjected to two rounds of directed evolution and the clones screened for increased stability and activity at pH 6.5. Beneficial mutation sites were investigated by semi-rational and combinatorial mutagenesis. Fourteen variants were characterised in detail to evaluate changes of the kinetic constants. Mutations increasing thermostability were distributed over the entire structure. Among them, T383I showed a 2.6-fold increased half-life by preventing the loss of the T2 copper through unfolding of a loop. Mutations affecting the pH-dependence cluster around the T1 copper and categorise in three types of altered pH profiles: pH-type I changes the monotonic decreasing pH profile into a bell-shaped profile, pH-type II describes increased specific activity below pH 6.5, and pH-type III increased specific activity above pH 6.5. Specific activities of the best variants were up to 5-fold higher (13 U mg -1 ) than BaL WT at pH 7.5.