Construction of Zr-Metal-Organic Frameworks-Based Composite Materials toward Anhydrous Proton Conduction and Photocatalytic CO2 Reduction.

Metal-organic frameworks constructed from Zr usually possess excellent chemical and physical stability. Therefore, they have become attractive platforms in various fields. In this work, two families of hybrid materials based on ZrSQU have been designed and synthesized, named Im@ZrSQU and Cu@ZrSQU, respectively. Im@ZrSQU was prepared through the impregnation method and employed for proton conduction. Im@ZrSQU exhibited terrific proton conduction performance in an anhydrous environment, with the highest proton conduction value of 3.6 × 10-2 S cm-1 at 110 °C. In addition, Cu@ZrSQU was synthesized via the photoinduction method for the photoreduction of CO2, which successfully promoted the conversion of CO2 into CO and achieved the CO generation rate of up to 12.4 µmol g-1 h-1. The photocatalytic performance of Cu@ZrSQU is derived from the synergistic effect of Cu NPs and ZrSQU. Based on an in-depth study and discussion toward ZrSQU, we provide a versatile platform with applications in the field of proton conduction and photocatalysis, which will guide researchers in their further studies.

Danggui Buxue decoction ameliorates mitochondrial biogenesis and cognitive deficits through upregulating histone H4 lysine 12 acetylation in APP/PS1 mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Danggui Buxue decoction (DBD) is a classic herbal decoction consisting of Astragali Radix (AR) and Angelica Sinensis Radix (ASR) with a 5:1 wt ratio, which can supplement 'blood' and 'qi' (vital energy) for the treatment of clinical diseases. According to Traditional Chinese Medicine (TCM) theory, dementia is induced by Blood deficiency and Qi weakness, which causes a decline in cognition. However, the underlying mechanisms of DBD improving cognition deficits in neurodegenerative disease are no clear. AIM OF THE STUDY: This study aims at revealing the underlying mechanisms of DBD plays a protective role in the cognitive deficits and pathology process of Alzheimer's disease (AD). MATERIALS AND METHODS: The APP/PS1 (Mo/HuAPP695swe/PS1-dE9) double transgenic mice were adopted as an experimental model of AD. Qualitative and quantitative analysis of 3 compounds in DBT was analyzed by HPLC. Morris water maze test, Golgi staining and electrophysiology assays were used to evaluate the effects of DBD on cognitive function and synaptic plasticity in APP/PS1 mice. Western blot, immunofluorescence and Thioflavin S staining were used for the pathological evaluation of AD. Monitoring the level of ATP, mitochondrial membrane potential, SOD and MDA to evaluate the mitochondrial function, and with the usage of qPCR and CHIP for the changes of histone post-translational modification. RESULTS: In the current study, we found that DBD could effectively attenuate memory impairments and enhance long-term potentiation (LTP) with concurrent increased expression of memory-associated proteins. DBD markedly decreased Aß accumulation in APP/PS1 mice by decreasing the phosphorylation of APP at the Thr668 level but not APP, PS1 or BACE1. Further studies demonstrated that DBD restored mitochondrial biogenesis deficits and mitochondrial dysfunction. Finally, the restored mitochondrial biogenesis and cognitive deficits are under HADC2-mediated histone H4 lysine 12 (H4K12) acetylation at the peroxisome proliferator-activated receptor-gamma coactivator 1α (PGC-1α) and N-methyl-D-aspartate receptor type 2B (GluN2B) promoters. CONCLUSIONS: These findings reveal that DBD could ameliorate mitochondrial biogenesis and cognitive deficits by improving H4K12 acetylation. DBD might be a promising complementary drug candidate for AD treatment.

Cation affinity purification of histidine-tagged proteins.

Protein purification is a basic technology in both biological research and industrial production, and efficient, convenient, economical, and environmentally friendly purification methods have always been pursued. In this study, it was found that alkaline earth metal cations (Mg2+, Ca2+) and alkali metal cations (Li+, Na+, K+) and even nonmetal cations (e.g., NH4+, imidazole, guanidine, arginine, lysine) can precipitate multi-histidine-tagged proteins (at least two tags in a whole protein) at low salts concentrations that are 1-3 orders of magnitude lower than salting-out, and precipitated proteins could be dissolved at moderate concentration of corresponding cation. Based on this finding, a novel cation affinity purification method was developed, which requires only three centrifugal separations to obtain highly purified protein with purification fold similar to that of immobilized metal affinity chromatography. The study also provides a possible explanation for unexpected protein precipitation and reminds researchers to consider the influence of cations on the experimental results. The interaction between histidine-tagged proteins and cations may also have broad application prospects. KEY POINTS: • Histidine-tagged proteins can be precipitated by low-concentrations common cations • A novel nonchromatographic protein purification method was developed • Purified protein can be obtained in pellet form by only three centrifugations.

A porous Ti-based metal-organic framework for CO2 photoreduction and imidazole-dependent anhydrous proton conduction.

The anhydrous proton conductivity of Im@IEF-11 resulting from the integration of imidazole and porous IEF-11 has been investigated, and the highest proton conductive value can reach up to 7.64 × 10-2 S cm-1. Furthermore, IEF-11 is also developed to reduce CO2 due to its reasonable structure and suitable energy band, and its CO formation rate is 31.86 µmol g-1 h-1.

Dual-Functional Hosts for Polysulfides Conversion and Lithium Plating/Stripping towards Lithium-Sulfur Full Cells.

The practical application of lithium-sulfur (Li-S) batteries is greatly hindered by the shuttle effect of dissolved polysulfides in the sulfur cathode and the severe dendritic growth in the lithium anode. Adopting one type of effective host with dual-functions including both inhibiting polysulfide dissolution and regulating Li plating/stripping, is recently an emerging research highlight in Li-S battery. This review focuses on such dual-functional hosts and systematically summarizes the recent research progress and application scenarios. Firstly, this review briefly describes the stubborn issues in Li-S battery operations and the sophisticated counter measurements over the challenges by dual-functional behaviors. Then, the latest advances on dual-functional hosts for both cathode and anode in Li-S full cells are catalogued as species, including metal chalcogenides, metal carbides, metal nitrides, heterostuctures, and the possible mechanisms during the process. Besides, we also outlined the theoretical calculation tools for the dual-functional host based on the first principles. Finally, several sound perspectives are also rationally proposed for fundamental research and practical development as guidelines.

Ag Nanoparticle-Modified Polyoxometalate-Based Metal-Organic Framework for Enhanced CO2 Photoreduction.

The photoreduction deposition method is employed to fabricate a family of silver nanoparticle (Ag NP)-modified polyoxometalate-based metal-organic framework (NENU-5) photocatalysts, named Ag/NENU-5. The title photocatalysts, Ag/NENU-5, can be used for the photocatalytic reduction of CO2 and are observed to efficiently reduce CO2 into CO, in which the highest reduction rate is 22.28 µmol g-1 h-1, 3 times greater than that of NENU-5. Photocatalytic reduction performances of CO2 have been extremely improved after the incorporation of Ag NPs as the cocatalyst. The enhancement of the photocatalytic reduction of CO2 has been attributed to the synergistic effects of Ag NPs and NENU-5, inhibiting the charge recombination during the photocatalytic process and increasing the reaction active sites. Furthermore, the influence of Ag NPs on the photocatalytic activity has also been investigated. The experimental results clearly reveal that the size of Ag NPs could exert a main effect on the photocatalytic activity, and the reasonable size of Ag NPs is able to enhance the photocatalytic reduction activity toward CO2 significantly.

Identification of a Genomic Instability-Related Long Noncoding RNA Prognostic Model in Colorectal Cancer Based on Bioinformatic Analysis.

Background: In recent years, a growing body of research has revealed that long noncoding RNAs (lncRNAs) participate in regulating genomic instability. Materials and Methods: We obtained RNA expression profiles, somatic mutation profiles, clinical information, and pathological features of colorectal cancer (CRC) from The Cancer Genome Atlas project. We divided the cohort into two groups based on mutation frequency and identified genomic instability-related lncRNAs (GI-lncRNAs) using R software. We further analyzed the function of identified GI-lncRNAs and established a prognostic model through Cox regression. Using the established prognostic model, we divided the cohort into the high- and low-risk groups and further verified the prognostic differences between the two groups as well as the predictive power of prognosis-related lncRNAs in the genomic instability of CRC. Results: We identified a total of 143 GI-lncRNAs that were differentially expressed between the higher mutation frequency group and the lower mutation frequency group. According to Kyoto Encyclopedia of Genes and Genomes pathway and Gene Ontology analyses, a series of cancer-associated terms were enriched. We further constructed a prognostic model that included five GI-lncRNAs (lncRNA PTPRD-AS1, lncRNA AC009237.14, lncRNA LINC00543, lncRNA AP003555.1, and lncRNA AL109615.3). We confirmed that the expression of the five GI-lncRNAs was associated with prognosis and the mutation of critical genes in the CRC patient cohort. Conclusions: The present research further confirmed the vital function of GI-lncRNAs in the genomic instability of CRC. The five GI-lncRNAs identified in our study are potential biomarkers and need to be studied in more depth.

Effect of SpyTag/SpyCatcher cyclization on stability and refolding of green fluorescent protein.

To study the effect of SpyTag/SpyCatcher cyclization on stability and refolding of protein, we constructed a cyclized green fluorescent protein (SRGFP) and its derivative to act as a linear structure control (L-SRGFP). SRGFP and L-SRGFP showed similar fluorescence characteristics to the wild-type GFP, while compared with GFP and L-SRGFP, the thermal stability and denaturation resistance of SRGFP were improved. The refolding efficiencies of these three denatured proteins were investigated under different pH, temperature and initial protein concentration conditions, and it was found that SRGFP was superior to GFP and L-SRGFP in terms of refolding yield and refolding speed. In the pH range of 8.0-8.5, SRGFP could basically recover all fluorescence, while GFP and L-SRGFP recovered only about 87.52% and 88.58%. When refolded at a high temperature (37 °C), SRGFP still recovered 85.27% of the fluorescence, whereas GFP and L-SRGFP recovered only around 69.43% and 68.45%. At a high initial protein concentration (5 mg/mL), the refolding yield of SRGFP was about 15% higher than that of both GFP and L-SRGFP. These results suggest that the introduction of SpyRing structure (head-to-tail cyclization via SpyTag and SpyCatcher) improved the protein's stability and facilitated the refolding of denatured protein.

Encapsulation of Nitrilase in Zeolitic Imidazolate Framework-90 to Improve Its Stability and Reusability.

In this study, nitrilase (Nit) was immobilized in zeolite imidazole framework-90 (ZIF-90) by one-pot biomimetic mineralization strategy. The structure, morphology and functional groups of ZIF-90 and immobilized enzyme Nit@ZIF-90 were characterized by scanning electron microscopy (SEM)/energy-dispersive X-ray spectroscopy (EDX), transmission electron microscopy (TEM), X-ray diffraction (XRD), thermogravimetric analysis (TGA) and Fourier transform infrared spectroscopy (FT-IR). Circular dichroism (CD) proved that the immobilized method of encapsulation in ZIF-90 could effectively maintain the intrinsic conformation of Nit. Meanwhile, the stability and reusability of Nit@ZIF-90 were systematically evaluated. Compared with the free enzyme, the thermal, pH and organic solvents stability of Nit@ZIF-90 were significantly increased. Further, Nit@ZIF-90 exhibited better reusability during the hydrolysis of acrylonitrile and retained 48.34% of the initial activity after 10 cycles. Besides, the Ni@ZIF-90 had preferable storage stability, which showed a high degree of residual activity (more than 64 %) after storage at 4 °C for 7 d. The improved stability and reusability of the Nit@ZIF-90 implied that it could be used as a potential effective biocatalyst for hydrolysis of nitrile compounds in industrial application.

A multifunctional anionic metal-organic framework for high proton conductivity and photoreduction of CO2 induced by cation exchange.

Metal-organic frameworks (MOFs) provide an ideal platform for loading various guests owing to their available space, and can be developed as a class of multifunctional materials. Herein, we cover the design and synthesis of two kinds of exchanged frameworks with multifunctional applications based on H3ImDC and In(NO3)3·2H2O through guest exchange inside the framework. The guest ammonium ion (NH4+) and [Ru(2,2'-bipyridine)3]2+ (Rubpy) are selected to exchange the dimethylammonium cation (Me2NH2+) encapsulated within In-MOF, giving birth to two kinds of new MOFs, named NH4+@In-MOF and Rubpy@In-MOF respectively. The proton conduction of NH4+@In-MOF and the CO2 photoreduction of Rubpy@In-MOF are investigated. Under different test conditions, the proton conductive behaviors of NH4+@In-MOF are evaluated and the best proton conductive value can reach up to 9.81 × 10-3 S cm-1. Compared to the original In-MOF, Rubpy@In-MOF exhibits a significantly enhanced CO2 photoreduction performance under a pure CO2 atmosphere. Furthermore, its photocatalytic activity is retained even under a 10% CO2 gas atmosphere, displaying a synergistic effect between Rubpy and In-MOF24 within Rubpy@In-MOF.

Orexin A peptidergic system: comparative sleep behavior, morphology and population in brains between wild type and Alzheimer's disease mice.

Sleep disturbance is common in patients with Alzheimer's disease (AD), and orexin A is a pivotal neurotransmitter for bidirectionally regulating the amyloid-ß (Aß) deposition of AD brain and poor sleep. In the present study, we examined the characteristic of sleep-wake architecture in APPswe/PSldE9 (APP/PS1) and Aß-treated mice using electroencephalogram (EEG) and electromyographic (EMG) analysis. We compared the expression of orexin A, distribution, and morphology of the corresponding orexin A-positive neurons using innovative methods including three-dimensional reconstruction and brain tissue clearing between wild type (WT) and APP/PS1 mice. Results from our study demonstrated that increased wakefulness and reduced NREM sleep were seen in APP/PS1 and Aß treated mice, while the expression of orexin A was significantly upregulated. Higher density and distribution of orexin A-positive neurons were seen in APP/PS1 mice, with a location of 1.06 mm-2.30 mm away from the anterior fontanelle compared to 1.34 mm-2.18 mm away from the anterior fontanelle in WT mice. These results suggested that the population and distribution of orexin A may play an important role in the progression of AD.

SpyTag/Catcher chemistry induces the formation of active inclusion bodies in E. coli.

SpyTag/Catcher chemistry is usually applied to engineer robust enzymes via head-to-tail cyclization using spontaneous intramolecular isopeptide bond formation. However, the SpyTag/Catcher induced intercellular protein assembly in vivo cannot be ignored. It was found that some active inclusion bodies had generated to different proportions in the expression of six SpyTag/Catcher labeled proteins (CatIBs-STCProtein). Some factors that may affect the formation of CatIBs-STCProtein were discussed, and the subunit quantities were found to be strongly positively related to the formation of protein aggregates. Approximately 85.44% of the activity of the octameric protein leucine dehydrogenase (LDH) was expressed in aggregates, while the activity of the monomeric protein green fluorescence protein (GFP) in aggregates was 12.51%. The results indicated that SpyTag/Catcher can be used to form protein aggregates in E. coli. To facilitate the advantages of CatIBs-STCProtein, we took the CatIBs-STCLDH as an example and further chemically cross-linked with glutaraldehyde to obtain novel cross-linked enzyme aggregates (CLEAs-CatIBs-STCLDH). CLEAs-CatIBs-STCLDH had good thermal stability and organic solvents stability, and its activity remained 51.03% after incubation at 60 °C for 100 mins. Moreover, the crosslinked CatIBs-STCLDH also showed superior stability over traditional CLEAs, and its activity remained 98.70% after 10 cycles of catalysis.

Integration of zirconium-based metal-organic framework with CdS for enhanced photocatalytic conversion of CO2 to CO.

It is a promising strategy to prepare composite photocatalysts based on MOFs and semiconductors for enhancing photocatalytic reduction of carbon dioxide (CO2). A family of binary composite photocatalysts (CdS@UiO-66-NH2) with different CdS contents have been designed and synthesized, which have been explored for photocatalytic reduction of CO2. CdS@UiO-66-NH2 can efficiently convert CO2 into CO under visible light irradiation via the solid-gas mode in the absence of sacrificial agents and photosensitizers. The generation rate of CO can reach up to 280.5 µmol g-1 h-1, which is 2.13-fold and 2.9-fold improvements over the pristine CdS and UiO-66-NH2, respectively, and the selectivity for CO is very high. Furthermore, this kind of photocatalysts can still maintain great photocatalytic activity in CO2/N2 mixed atmosphere with different CO2 concentrations. The outstanding performances of CdS@UiO-66-NH2 may be attributed to the existence of the direct Z-scheme heterojunction, which possesses the enhanced separation and migration of photo-generated charge carriers between UiO-66-NH2 and CdS, available specific surface areas and improved visible light absorption ability as well as abundant reaction active sites. This case reveals that MOF-based composite photocatalysts exhibit promising potential applications in the field of CO2 conversion.

Dehydrocostus lactone inhibits BLM-induced pulmonary fibrosis and inflammation in mice via the JNK and p38 MAPK-mediated NF-κB signaling pathways.

Idiopathic pulmonary fibrosis (IPF) is a chronic and irreversible inflammatory disease with a high mortality rate and limited therapeutic options. This study explored the potential role and mechanisms of Dehydrocostus lactone (DHL) in the inflammatory and fibrotic responses in a bleomycin (BLM) induced model. Treatment with DHL significantly reduced pathological injury and fibrosis, the secretion of BLM-induced pro-fibrotic mediators TGF-ß and α-SMA, and components of the extracellular matrix (fibronectin). Additionally, in the early stages of inflammation, DHL administration inhibited the infiltration of inflammatory cells and downregulated the expression of TGF-ß, TNF-α, and IL-6, indicating that DHL treatment effectively alleviated BLM-induced pulmonary fibrosis and inflammation in a dose-dependent manner. Furthermore, BLM induced the production of IL-33 in vivo, which initiated and progressed pulmonary fibrosis by activating macrophages and enhancing the production of IL-13 and TGF-ß. In contrast, a significant decrease in the expression of IL-33 after DHL treatment in vitro showed that DHL strongly reduced IL-13 and TGF-ß. Regarding the mechanism, BLM-induced phosphorylation of JNK, p38 MAPK, and NF-κB were significantly reduced after DHL treatment, which further led to the down-regulation of IL-33 expression, thereby decreasing IL-13 and TGF-ß. Collectively, our data suggested that DHL could exert its anti-fibrosis effect via inhibiting the early inflammatory response by downregulating the JNK/p38 MAPK-mediated NF-κB signaling pathway to suppress macrophage activation. Therefore, DHL has therapeutic potential for pulmonary fibrosis.

One-step purification and immobilization of recombinant proteins using SpyTag/SpyCatcher chemistry.

Based on the specific and spontaneous formation of isopeptide bonds by SpyCatcher/SpyTag, we have developed a one-step method for purification and immobilization of recombinant proteins. The procedure is to immobilize SpyCatcher on glyoxyl agarose gels, and then the SpyCatcher immobilisate can be used to immobilize the SpyTag-fused protein in the crude extract selectively. A mutant of SpyCatcher (mSC), in which a peptide (LysGlyLysGlyLysGly) was added to the C-terminus of SpyCatcher and three lysine residues around the SpyTag/SpyCatcher binding domain were replaced with arginine, was designed to improve the attachment of SpyCatcher to the support. Compared with wild-type SpyCatcher, mSC can be immobilized on the glyoxyl-agarose support more efficiently, which enables the obtained mSC derivative a high binding capacity of the SpyTag-fused protein. The results showed that the target proteins in the crude enzyme extract were purified and immobilized in one step, and the thermal stability of the immobilized target proteins was also remarkably improved.

Optimization of Cephalosporin C Acylase Expression in Escherichia coli by High-Throughput Screening a Constitutive Promoter Mutant library.

Cephalosporin C acylase (CCA) is capable of catalyzing cephalosporin C (CPC) to produce 7-aminocephalosporanic acid (7-ACA), an intermediate of semi-synthetic cephalosporins. Inducible expression is usually used for CCA. To improve the efficiency of CCA expression without gene induction, three recombinant strains regulated by constitutive promoters BBa_J23105, PLtetO1, and tac were constructed, respectively. Among them, BBa_J23105 was the best promoter and its mutant libraries were established using saturation mutagenesis. In order to obtain the mutants with enhanced activity, a high-throughput screening method based on flow cytometric sorting techniques was developed by using green fluorescent protein (GFP) as the reporter gene. A series of mutants were screened at 28 °C, 200 rpm, and 24-h culture condition. The study of mutants showed that the enzyme activity, fluorescence intensity, and promoter transcriptional strength were positively correlated. The enzyme activity of the optimal mutant obtained by screening reached 12772 U/L, 3.47 times that of the original strain.

Self-assembly of TiO2/ZIF-8 nanocomposites for varied photocatalytic CO2 reduction with H2O vapor induced by different synthetic methods.

Photoreduction of carbon dioxide (CO2) provides an effective perspective for solving the energy crisis and environmental problems. Herein, two types of composite photocatalysts (TiO2/ZIF-8) based on ZIF-8 and TiO2 have been designed and synthesized with the help of the grinding method and the solid-synthesis method. Both composite photocatalysts are employed for the photocatalytic reduction of CO2. In composite photocatalysts prepared by the grinding method, ZIF-8 particles are distributed on the surface of TiO2, and provide extra available spaces for storing CO2, which is beneficial for improving their photoreduction performances. As a result, an enhanced CO formation rate of 21.74 µmol g-1 h-1 with a high selectivity of 99% is obtained for this family of composite photocatalysts via the solid-gas mode without photosensitizers and sacrificial agents. For comparison, the other family of composite photocatalysts synthesized via the solid-synthesis method possesses structures similar to ZIF-8, where TiO2 is encapsulated inside the framework of ZIF-8. This structural feature obstructs the contact between the active sites of TiO2 and CO2, and leads to lower activities. The best CO formation rate of this family is only 10.67 µmol g-1 h-1 with 90% selectivity. Both the structural features of the two families of photocatalysts are described to explain their differences in photoreduction performances. The experimental finding reveals that different synthetic approaches indeed result in diversified structures and varied photocatalytic performances. This work affords a new scalable and efficient approach for the rational design of efficient photocatalysts in the area of artificial photosynthesis.

Sophoricoside attenuates lipopolysaccharide-induced acute lung injury by activating the AMPK/Nrf2 signaling axis.

Sophoricoside (SOP), an isoflavone glycoside isolated from seed of Sophora japonica L., has been reported to have various pharmacological activities, including anti-cancer, anti-allergy and anti-inflammation. However, the effect of SOP on lipopolysaccharides (LPS)-acute lung injury (ALI) is completely unclear. Here, we found that SOP pretreatment significantly ameliorated LPS-induced pathological damage, tissue permeability, neutrophil infiltration and the production of pro-inflammatory cytokines (TNF-α, IL-1ß and IL-6) in a murine model of ALI. Besides, SOP reduced the production of pro-inflammatory mediators such as iNOS, NO and inflammatory cytokines including TNF-α, IL-1ß and IL-6 in LPS-stimulated RAW264.7 cells and bone marrow derived macrophages. Interestingly, treatment with SOP exhibited no effect on the activation of NF-κB and MAPKs in macrophages but prominently accelerated the expression and nuclear translocation of Nrf2. By using ML385, a specific Nrf2 inhibitor, we found that inhibition of Nrf2 abolished the inhibitory effect of SOP on LPS-induced iNOS expression, NO production as well as pro-inflammatory cytokine generation. SOP also activated AMPK, an upstream protein of Nrf2, under LPS stimuli. Furthermore, we demonstrated that the accelerated expression of Nrf2 induced by SOP was reversed by interference with the AMPK inhibitor Compound C. Taken together, our results suggested that SOP attenuated LPS-induced ALI in AMPK/Nrf2 dependent manner and indicated that SOP might be a potential therapeutic candidate for treating ALI/ARDS.

Effect of shaking speed on immobilization of cephalosporin C acylase: Correlation between protein distribution and properties of the immobilized enzymes.

During enzyme immobilization, enzyme activity and protein distribution are affected by various factors such as enzyme load, temperature, and pH. In general, two types of protein distribution patterns (heterogeneous or homogeneous) are observed inside a porous carrier, owing to differences in preparation parameters. During the immobilization of a fusion protein (CCApH) of cephalosporin C acylase (CCA) and pHluorin (a pH-sensitive mutant of green fluorescent protein), different shaking speeds induced obvious differences in protein distribution on an epoxy carrier, LX-1000EPC. Enzyme immobilization with a homogeneous distribution pattern was observed at a low shaking speed (120 rpm) with an operational stability of 10 batches at 37°C. The operational stability of an immobilisate with heterogeneous protein distribution prepared at a high shaking speed (200 rpm) was six batches. Given the pH-sensitive characteristics of pHluorin in the fusion protein, the intraparticle pH of CCApH immobilisates during catalysis was monitored using confocal laser scanning microscopy. The microenvironmental pH of the immobilisate with heterogeneous protein distribution sharply decreased by about 2 units; this decrease in the pH may be detrimental to the life-span of immobilized CCA. Thus, this work demonstrates the good operational stability of pH-sensitive proton-forming immobilized enzymes with homogeneous protein distribution.