The amounts and ratio of nitrogen and phosphorus addition drive the rate of litter decomposition in a subtropical forest.

Nitrogen (N) and phosphorus (P) control biogeochemical cycling in terrestrial ecosystems. However, N and P addition effects on litter decomposition, especially biological pathways in subtropical forests, remain unclear. Here, a two-year field litterbag experiment was employed in a subtropical forest in southwestern China to examine N and P addition effects on litter biological decomposition with nine treatments: low and high N- and P-only addition (LN, HN, LP, and HP), NP coaddition (LNLP, LNHP, HNLP, and HNHP), and a control (CK). The results showed that the decomposition coefficient (k) was higher in NP coaddition treatments (P < 0.05), and lower in N- and P-only addition treatments than in CK (P < 0.05). The highest k was observed with LNLP (P < 0.05). The N- and P-only addition treatments decreased the losses of litter mass, lignin, cellulose, and condensed tannins, litter microbial biomass carbon (MBC), litter cellulase, and soil pH (P < 0.05). The NP coaddition treatments increased the losses of litter mass, lignin, and cellulose, MBC concentration, litter invertase, urease, cellulase, and catalase activities, soil arthropod diversity (S) in litterbags, and soil pH (P < 0.05). Litter acid phosphatase activity and N:P ratio were lower in N-only addition treatments but higher in P-only addition and NP coaddition treatments than in CK (P < 0.05). Structural equation model showed that litter MBC, S, cellulase, acid phosphatase, and polyphenol oxidase contributed to the loss of litter mass (P < 0.05). The litter N:P ratio was negatively logarithmically correlated with mass loss (P < 0.01). In conclusion, the negative effect of N addition on litter decomposition was reversed when P was added by increasing decomposed litter soil arthropod diversity, MBC concentration, and invertase and cellulase activities. Finally, the results highlighted the important role of the N:P ratio in litter decomposition.

The responses of soil bacterial communities and enzyme activities to the edaphic properties of coal mining areas in Central China.

Soil physicochemical properties, bacterial communities and enzyme activities change with land subsidence resulting from coal mining. However, research on the responses of bacterial communities and enzyme activities to the soil properties in different degree of subsidence areas is limited. As such, we collected soil samples from a control area (C area), a moderate mining subsidence area (M area) and a severe mining subsidence area (S area) in Central China. Soil properties, such as the pH, total nitrogen (TN) content, total phosphorus (TP) content, available phosphorus (AP) content, organic matter (OM) content, and soil enzyme (urease, invertase, catalase and alkaline phosphatase) activities were measured in each sampling area at depths of 0-20 cm, 20-40 cm, and 40-60 cm. The results indicated that the soil physiochemical properties, soil urease activity, soil alkaline phosphatase activity and soil bacterial richness and diversity in the topsoil (0-20 cm) of the mining subsidence area were significantly lower than those in the C area. However, the soil enzyme activities within the deepest layer of the subsidence area were significantly greater than those of the C area. The bacterial communities within the depth of 0-20 cm were dominated by RB41, Pseudomonas, MND1, Nitrospira, Trichococcus, Sphingomonas and Dongia, whereas RB41 and Pseudomonas were the dominant species in the C area and subsidence area, respectively. Using correlation analysis, we found that the soil pH value, soil AP content and activities of the four enzymes were the main factors affecting the soil bacterial community structure. In addition, the soil nutrient contents, enzyme activities and bacterial richness and evenness decreased with increasing subsidence degree (classified by geological hazards, groundwater and landscape damage degree of coal mining subsidence). These results provide a reliable basis for environmental management of mining areas.

The sensitivity of soil microbial respiration declined due to crop straw addition but did not depend on the type of crop straw.

An incubation experiment was conducted to investigate whether the type of crop straw added to soil influenced the temperature sensitivity of soil microbial respiration. The soil for incubation was collected from a winter wheat-soybean rotation cropland. Five temperature levels (5, 10, 15, 20, and 25 °C), five crop straw types (soybean, peanut, rice, winter wheat, and maize), and a control (CK, no crop straw addition) were established. Soil microbial respiration rates were measured on days 1, 2, 3, 5, 7, 10, 14, 20, and 27 after crop straw addition using an infrared CO2 analyser. Soil enzyme activities of invertase, urea, and catalase and the dissolved organic carbon (DOC) content were measured after incubation. Estimated Q10 (temperature sensitivity of soil microbial respiration) ranged from 1.472 ± 0.045 to 1.970 ± 0.020 and showed no significant (P > 0.05) difference between straw addition treatments, but there was significantly (P < 0.001) higher temperature sensitivity (1.970 ± 0.020) for CK. A significant (P = 0.002) relationship was found between the Q10 of cumulative soil microbial respiration and basal soil microbial respiration (soil microbial respiration at 0 °C). Moreover, a marginally significant (P < 0.1) relationship was found between the Q10 at different incubation stages and basal soil microbial respiration. A quadratic function was used to explain the relationship between estimated basal microbial respiration and the lignin content. Soil microbial respiration was positively correlated with the activities of invertase, urease, and catalase and the dissolved organic carbon (DOC) content in all treatments. This study indicated that crop straw addition significantly (P < 0.001) reduced the Q10 of soil microbial respiration and that the types of crop straw added to soil did not significantly (P > 0.05) change the Q10 value.

Changes in sugar content and related enzyme activities in table grape (Vitis vinifera L.) in response to foliar selenium fertilizer.

BACKGROUND: Spraying selenium (Se) fertilizer is an effective method for Se-enriched fruit production. Sugar content in fruit is the major factor determining berry quality. However, changes in sugar metabolism in response to Se fertilizer are unclear. Hence, this study was conducted to identify the effects of Se fertilizer on sugar metabolism and related enzyme activities of grape berries. Additionally, production of leaves with and without Se fertilizer was also investigated. RESULTS: Acid invertase (AI) activity, total soluble sugar and Se content in berries, and photosynthetic rate in leaves produced under Se fertilizer treatments were higher than that of control. Glucose and fructose were the primary sugars in berries, with a trace of sucrose. In both berries and leaves, neutral invertase activity was lower than AI, there was no significant difference in neutral invertase, sucrose synthase and sucrose phosphate synthase between Se fertilizer-treated and control. In berries, AI showed a significant positive correlation with glucose and fructose; also Se content was significantly correlated with sugar content. CONCLUSION: AI played an important role in the process of sugar accumulation in berries; high AI activity in berries and photosynthetic rate in leaves could explain the mechanism by which Se fertilizer affected sugar accumulation in berries. © 2017 Society of Chemical Industry.

Establishing the relative importance of damaged starch and fructan as sources of fermentable sugars in wheat flour and whole meal bread dough fermentations.

It is generally believed that maltose drives yeast-mediated bread dough fermentation. The relative importance of fructose and glucose, released from wheat fructan and sucrose by invertase, compared to maltose is, however, not documented. This is surprising given the preference of yeast for glucose and fructose over maltose. This study revealed that, after 2h fermentation of wheat flour dough, about 44% of the sugars consumed were generated by invertase-mediated degradation of fructan, raffinose and sucrose. The other 56% were generated by amylases. In whole meal dough, 70% of the sugars consumed were released by invertase activity. Invertase-mediated sugar release seems to be crucial during the first hour of fermentation, while amylase-mediated sugar release was predominant in the later stages of fermentation, which explains why higher amylolytic activity prolonged the productive fermentation time only. These results illustrate the importance of wheat fructan and sucrose content and their degradation for dough fermentations.

Seasonal Dynamics of Soil Labile Organic Carbon and Enzyme Activities in Relation to Vegetation Types in Hangzhou Bay Tidal Flat Wetland.

Soil labile organic carbon and soil enzymes play important roles in the carbon cycle of coastal wetlands that have high organic carbon accumulation rates. Soils under three vegetations (Phragmites australis, Spartina alterniflora, and Scirpusm mariqueter) as well as bare mudflat in Hangzhou Bay wetland of China were collected seasonally. Seasonal dynamics and correlations of soil labile organic carbon fractions and soil enzyme activities were analyzed. The results showed that there were significant differences among vegetation types in the contents of soil organic carbon (SOC) and dissolved organic carbon (DOC), excepting for that of microbial biomass carbon (MBC). The P. australis soil was with the highest content of both SOC (7.86 g kg-1) and DOC (306 mg kg-1), while the S. mariqueter soil was with the lowest content of SOC (6.83 g kg-1), and the bare mudflat was with the lowest content of DOC (270 mg kg-1). Soil enzyme activities were significantly different among vegetation types except for urease. The P. australis had the highest annual average activity of alkaline phosphomonoesterase (21.4 mg kg-1 h-1), and the S. alterniflora had the highest annual average activities of ß-glycosidase (4.10 mg kg-1 h-1) and invertase (9.81 mg g-1 24h-1); however, the bare mudflat had the lowest activities of alkaline phosphomonoesterase (16.2 mg kg-1 h-1), ß-glycosidase (2.87 mg kg-1 h-1), and invertase (8.02 mg g-1 24h-1). Analysis also showed that the soil labile organic carbon fractions and soil enzyme activities had distinct seasonal dynamics. In addition, the soil MBC content was significantly correlated with the activities of urease and ß-glucosidase. The DOC content was significantly correlated with the activities of urease, alkaline phosphomonoesterase, and invertase. The results indicated that vegetation type is an important factor influencing the spatial-temporal variation of soil enzyme activities and labile organic carbon in coastal wetlands.

Direct, simultaneous quantification of fructooligosaccharides by FT-MIR ATR spectroscopy and chemometrics for rapid identification of superior, engineered ß-fructofuranosidases.

Fructooligosaccharides (FOS) are popular components of functional foods produced by the enzymatic transfer of fructose units to sucrose. Improving ß-fructofuranosidase traits by protein engineering is restricted by the absence of a rapid, direct screening method for the fructooligosaccharide products produced by enzyme variants. The use of standard high-performance liquid chromatography (HPLC) methods involves time-consuming sample preparation and chromatographic and data analysis steps. To overcome these limitations, this work presents a rapid method for screening ß-fructofuranosidase variant libraries using Fourier transform mid-infrared attenuated total reflectance (FT-MIR ATR) spectroscopy and calibration using partial least squares (PLS) regression. The method offers notable improvements in terms of sample analysis times and cost, with the added benefit of the absence of toxic eluents. Wavenumber interval selection methods were tested to develop optimised PLS regression models that were successfully applied to quantify of glucose, fructose, sucrose, 1-kestose and nystose, the substrates and products of ß-fructofuranosidase activity. To the best of our knowledge, this is the first report on the use of infrared spectroscopy and PLS calibration for the quantification of 1-kestose and nystose. Independent test set-validated results indicated that optimal wavenumber selection by interval PLS (iPLS) served to provide the best models for all sugars, bar glucose. Application of this screening method will facilitate the engineering of ß-fructofuranosidases and other glycosyltransferase enzymes by random mutagenesis strategies, as it provides, for the first time, a rapid, direct assay for transferase products that may be adapted to a high-throughput set-up.

Long-term effects of fertilizer on soil enzymatic activity of wheat field soil in Loess Plateau, China.

The effects of long-term (29 years) fertilization on local agro-ecosystems in the Loess Plateau of northwest China, containing a single or combinations of inorganic (Nitrogen, N; Phosphate, P) and organic (Mature, M Straw, S) fertilizer, including N, NP, SNP, M, MNP, and a control. The soil enzymes, including dehydrogenase, urease, alkaline phosphatase, invertase and glomalin, were investigated in three physiological stages (Jointing, Dough, and Maturity) of wheat growth at three depths of the soil profile (0-15, 16-30, 31-45 cm). We found that the application of farmyard manure and straw produced the highest values of soil enzymatic activity, especially a balanced applied treatment of MNP. Enzymatic activity was lowest in the control. Values were generally highest at dough, followed by the jointing and maturity stages, and declined with soil profile depth. The activities of the enzymes investigated here are significantly correlated with each other and are correlated with soil nutrients, in particular with soil organic carbon. Our results suggest that a balanced application of fertilizer nutrients and organic manure (especially those containing P) has positive effects on multiple soil chemical parameters, which in turn enhances enzyme activity. We emphasize the role of organic manure in maintaining soil organic matter and promoting biological activity, as its application can result in a substantial increase in agricultural production and can be sustainable for many years.

Effect of repeated applications of buprofezin and acephate on soil cellulases, amylase, and invertase.

The impact of repeated applications of buprofezin and acephate, at concentrations ranging from 0.25 to 1.0 kg ha(-1), on activities of cellulases, amylase, and invertase in unamended and nitrogen, phosphorous, and potassium (NPK) fertilizer-amended soil planted with cotton was studied. The nontarget effect of selected insecticides, when applied once, twice, or thrice on soil enzyme activities, was dose-dependent; the activities decreased with increasing concentrations of insecticides. However, there was a rapid decline in activities of enzymes after three repeated applications of insecticides in unamended or NPK-amended soil. Our data clearly suggest that insecticides must be applied judiciously in pest management in order to protect the enzymes largely implicated in soil fertility.

Using the widely available blood glucose meter to monitor insulin and HbA1c.

The blood glucose meter (BGM) is the most successful and widely used portable device for point-of-care (POC) tests. However, its usage is limited to self-monitoring of blood glucose level only. To expand the targets that BGM can monitor while taking advantage of more than 50 years of technology development, we report herein the use of BGM to detect and quantify insulin and glycated hemoglobin (HbA1c), which are useful hormone for diabetes treatment and biomarker for diabetes monitoring, respectively. The method is based on invertase enzyme-linked immunosorbent assay (iELISA) and phosphatase enzyme-linked immunosorbent assay (pELISA) that convert BGM-inert sucrose or glucose-1-phosphate into glucose in the presence of insulin and glycated hemoglobin, respectively. In both assays, monoclonal antibodies specific to the targets (insulin or HbA1c) are immobilized onto magnetic beads to capture the targets in samples, followed by the formation of sandwich complex with the polyclonal antibodies conjugated to either invertase or phosphatase. The quantification of the targets is then realized by the production of glucose from the biochemical reactions catalyzed by the polyclonal antibody-enzyme conjugates bound on the surface of the magnetic beads. Such a method can be generally applied to a wide range of other biomarkers using the corresponding antibodies.

Effect of vegetation of transgenic Bt rice lines and their straw amendment on soil enzymes, respiration, functional diversity and community structure of soil microorganisms under field conditions.

With the development of transgenic crops, there is an increasing concern about the possible adverse effects of their vegetation and residues on soil environmental quality. This study was carried out to evaluate the possible effects of the vegetation of transgenic Bt rice lines Huachi B6 (HC) and TT51 (TT) followed by the return of their straw to the soil on soil enzymes (catalase, urease, neutral phosphatase and invertase), anaerobic respiration activity, microbial utilization of carbon substrates and community structure, under field conditions. The results indicated that the vegetation of the two transgenic rice lines (HC and TT) and return of their straw had few adverse effects on soil enzymes and anaerobic respiration activity compared to their parent and distant parent, although some transient differences were observed. The vegetation and subsequent straw amendment of Bt rice HC and TT did not appear to have a harmful effect on the richness, evenness and community structure of soil microorganisms. No different pattern of impact due to plant species was found between HC and TT. It could be concluded that the vegetation of transgenic Bt rice lines and the return of their straw as organic fertilizer may not alter soil microbe-mediated functions.

Improved enzyme production by bio-pellets of Aspergillus niger: targeted morphology engineering using titanate microparticles.

The present study describes the design of bio-pellet morphologies of the industrial working horse Aspergillus niger strains in submerged culture. The novel approach recruits the intended addition of titanate microparticles (TiSiO(4), 8 µm) to the growth medium. As tested for two recombinant strains producing fructofuranosidase and glucoamylase, the enzyme titer by the titanate-enhanced cultures in shake flasks was increased 3.7-fold to 150 U/mL (for fructofuranosidase) and 9.5-fold to 190 U/mL (for glucoamylase) as compared to the control. This could be successfully utilized for improved enzyme production in stirred tank reactors. Stimulated by the particles, the achieved final glucoamylase activity of 1,080 U/mL (fed-batch) and 320 U/mL (batch) was sevenfold higher as compared to the conventional processes. The major reason for the enhanced production was the close association between the titanate particles and the fungal cells. Already below 2.5 g/L the micromaterial was found inside the pellets, including single particles embedded as 50-150 µm particle aggregates in the center resulting in core shell pellets. With increasing titanate levels the pellet size decreased from 1,700 µm (control) to 300 µm. Fluorescence based resolution of GFP expression revealed that the large pellets of the control were only active in a 200 µm surface layer. This matches with the critical penetration depth for nutrients and oxygen typically observed for fungal pellets. The biomass within the titanate derived fungal pellets, however, was completely active. This was due a reduced thickness of the biomass layer via smaller pellets as well as the core shell structure. Moreover, also the created loose inner pellet structure enabled a higher mass transfer and penetration depths for up to 500 µm. The creation of core-shell pellets has not been achieved previously by the addition of microparticles, for example, made of talc or alumina. Due to this, the present work opens further possibilities to use microparticles for tailor-made morphology design of filamentous fungi, especially for pellet based processes which have a long and strong industrial relevance for industrial production.

Dissipation of carbendazim and chloramphenicol alone and in combination and their effects on soil fungal:bacterial ratios and soil enzyme activities.

The dissipation of carbendazim and chloramphenicol alone and in combination and their effects on soil fungal:bacterial ratios and soil enzyme activities were investigated. The results revealed that carbendazim dissipation was little affected by chloramphenicol, whereas chloramphenicol dissipation was found to be retarded significantly by the presence of carbendazim. The inhibitory effect of carbendazim on the fungal:bacterial ratios was increased by the presence of chloramphenicol, and the inhibitory effect of chloramphenicol on neutral phosphatase was increased by the presence of carbendazim. Carbendazim increased soil catalase and urease activities, but this increase was partially diminished by the presence of chloramphenicol. Little interaction was observed between carbendazim and chloramphenicol with regard to their influence on soil invertase. The results obtained in this study suggest that combinations of fungicides and antibiotics may alter the compounds' individual behaviors in soil and their effects on soil enzymes.

[Effects of long-term fertilization on soil enzyme activities and soil physicochemical properties of facility vegetable field].

An investigation was made on a long-term fertilization facility vegetable field at Shenyang Agricultural University to study the effects of long-term fertilization on the soil enzyme activities and soil physicochemical properties. Long term application of organic manure combined with or without nitrogen fertilizer increased the contents of soil organic matter, N, P, and K, and improved the soil physical properties and soil invertase, urease, and neutral phosphatase activities. However, long-term application of nitrogen fertilizer alone decreased soil pH and soil enzymes activities. Significant positive correlations were observed between soil invertase activity and soil organic matter and total P, between soil urease activity and soil organic matter, alkali-hydrolyzable N, total and available P, and available K, and between soil neutral phosphatase activity and soil organic matter, total P, and available K, but less correlation was found between soil dehydrogenase activity and soil nutrients.

Grape and wine proteins: their fractionation by hydrophobic interaction chromatography and identification by chromatographic and proteomic analysis.

A method to fractionate grape and wine proteins by hydrophobic interaction chromatography (HIC) was developed. This method allowed the isolation of a thaumatin-like protein in a single step with high yield and >90% purity and has potential to purify several other proteins. In addition, by separating HIC fractions by reverse phase HPLC and by collecting the obtained peaks, the grape juice proteins were further separated, by SDS-PAGE, into 24 bands. The bands were subjected to nanoLC-MS/MS analysis, and the results were matched against a database and characterized as various Vitis vinifera proteins. Moreover, either directly or by homology searching, identity or function was attributed to all of the gel bands identified, which mainly consisted of grape chitinases and thaumatin-like proteins but also included vacuolar invertase, PR-4 type proteins, and a lipid transfer protein from grapes.

Acid invertase localization in leaves of the fern Pteris deflexa link.

The localization of invertase, a key enzyme in plant carbohydrate metabolism, has been established in several higher plants, but there are no reports of it in ferns. The aim of the present work was to establish the localization of the previously reported acid invertase activity of Pteris deflexa in fronds tissues and to compare the findings with invertase localization in higher plants. Acid invertase, localized by immuno-histochemical and histochemical techniques on fresh tissues, was evident in vascular tissue, mainly in phloem. It was also detected in parenchymatic, sclerenchymatic and epidermic cells of petiole, rachis and rachis branches as well as in veins of leaf blades. Our results demonstrate that P. deflexa acid invertase localization is the same to that of higher plants. Hence, potential roles of the fern enzyme in relation to the storage and utilization of sucrose and to control carbon flux could be the same of those proposed to higher plants.

[Spatiotemporal evolvement of soil microbiological characteristics in upland fields with different utilization duration in Cixi, Zhejiang Province].

The microbial number, microbial biomass, and enzymatic activities in five upland soils under agricultural utilization for 50-700 years were determined, with the correlations between soil microbiological characteristics and agricultural utilization duration analyzed. In the meantime, the functional diversity of microbes in soils having been utilized for 50, 100, and 700 years were investigated. The results showed that at the early stage (< 100 years) of agricultural utilization, the number of soil fungi (F) had a slight increase, while the bacterial number (B), B/F ratio, microbial biomass C (C(mic)), microbial biomass N (N(mic)), and the activities of catalase, invertase and urease all decreased markedly. After utilized for more than 100 years, the F decreased significantly, while the B, B/F ratio, C(mic), N(mic), and the activities of test enzymes all tended to increase. During the whole utilization period from 50 to 700 years, the C(mic)/N(mic) ratio had a significant increase with year. The Shannon, Simpson, and McIntosh indices of soil microbial community had the same responses to the agricultural utilization duration as the bacterial number, microbial biomass, and enzymatic activities. All of these indicated that in the upland fields in Cixi of Zhejiang Province, shifts of soil microbial community occurred with increasing agricultural utilization duration, and soil microbiological quality had an overall increase after 100 years agricultural utilization.

Stimulation of zero-trans rates of lactose and maltose uptake into yeasts by preincubation with hexose to increase the adenylate energy charge.

Initial rates of sugar uptake (zero-trans rates) are often measured by incubating yeast cells with radiolabeled sugars for 5 to 30 s and determining the radioactivity entering the cells. The yeast cells used are usually harvested from growth medium, washed, suspended in nutrient-free buffer, and stored on ice before they are assayed. With this method, the specific rates of zero-trans lactose uptake by Kluyveromyces lactis or recombinant Saccharomyces cerevisiae strains harvested from lactose fermentations were three- to eightfold lower than the specific rates of lactose consumption during fermentation. No significant extracellular beta-galactosidase activity was detected. The ATP content and adenylate energy charge (EC) of the yeasts were relatively low before the [(14)C]lactose uptake reactions were started. A short (1- to 7-min) preincubation of the yeasts with 10 to 30 mM glucose caused 1.5- to 5-fold increases in the specific rates of lactose uptake. These increases correlated with increases in EC (from 0.6 to 0.9) and ATP (from 4 to 8 micromol x g dry yeast(-1)). Stimulation by glucose affected the transport V(max) values, with smaller increases in K(m) values. Similar observations were made for maltose transport, using a brewer's yeast. These findings suggest that the electrochemical proton potential that drives transport through sugar/H(+) symports is significantly lower in the starved yeast suspensions used for zero-trans assays than in actively metabolizing cells. Zero-trans assays with such starved yeast preparations can produce results that seriously underestimate the capacity of sugar/H(+) symports. A short exposure to glucose allows a closer approach to the sugar/H(+) symport capacity of actively metabolizing cells.

Purification of alpha-galactosidase and invertase by three-phase partitioning from crude extract of Aspergillus oryzae.

Alpha-galactosidase and invertase were accumulated in a coherent middle phase in a three-phase partitioning system under different conditions (ammonium sulphate, ratio of tert-butanol to crude extract, temperature and pH). Alpha-galactosidase and invertase were purified 15- and 12-fold with 50 and 54% activity recovery, respectively. The fractions of interfacial precipitate arising from the three-phase partitioning were analyzed by SDS-PAGE. Both purified preparations showed electrophoretic homogeneity on SDS-PAGE.

Structure/function relationships of several biopolymers as related to invertase stability in dehydrated systems.

Structure/function relationships of different biopolymers (alginate, dextran, or beta-cyclodextrin) were analyzed as single excipients or combined with trehalose in relation to their efficiency as enzyme stabilizers in freeze-dried formulations and compared to trehalose. Particularly, a novel synthesized polymer beta-cyclodextrin-branched alginate (beta-CD-A) was employed as excipient. During freeze-drying, the polymers or their mixtures did not confer better protection to invertase compared to trehalose. Beta-CD-A (with or without trehalose), beta-cyclodextrin (beta-CD), or dextran with trehalose were the best protective agents during thermal treatment, while beta-CD and alginate showed a negative effect on invertase activity preservation. The beta-CD linked alginate combined the physical stability provided by alginate with the stabilization of hydrophobic regions of the enzyme provided by cyclodextrin. Beta-CD-A was effective even at conditions at which trehalose lost its protective effect. A relatively simple covalent combination of two biopolymers significantly affected their functionalities and, consequently, their interactions with proteins, modifying enzyme stability patterns.