Mechanism of solvent induced thermal stabilization of papain

In the present study an attempt is made to elucidate the effects of various cosolvents, such as sorbitol, sucrose, xylose and glycerol, on papain. The stabilizing effects of these cosolvents on the structure and function of papain is determined by the activity measurements, fluorescence spectroscopy and differential scanning calorimetry (DSC). The enzyme activity measurements indicate several fold increase in the thermal stability of the enzyme in all the cosolvents used. The thermal denaturation studies of papain in presence of various concentrations of cosolvents indicated a shift in the apparent thermal denaturation temperature (app Tm) suggesting increased thermal stability of papain in presence of cosolvents. The app Tm shifted from a control value of 83+/-1 degrees C to a value of >90+/-1 degrees C in presence of 40% sorbitol. The DSC thermogram for native papain can be clearly deconvoluted into two transitions corresponding to left and right domain and in presence of cosolvents both transitions A and B shift to higher temperature. Maximum stabilization was seen in case of 30% sorbitol where the thermal transition temperatures increased compared to control. The results from partial specific volume measurements of papain in presence of cosolvents suggest that the preferential interaction parameter (xi3) was negative in all cosolvents and maximum hydration was observed in the case of glycerol where the preferential interaction parameter was 0.165g/g. These above results suggest that there is a considerable increase in the thermal stability of papain in presence of these cosolvents as a result of preferential hydration.     

High-performance affinity chromatography for characterization of human immunoglobulin G digestion with papain

Reactive continuous rods of macroporous poly(glycidyl methacrylate-co-ethylene dimethacrylate) were prepared within the confines of a stainless steel column. Then papain was immobilized on these monoliths either directly or linked by a spacer arm. In a further step, a protein A affinity column was used for the characterization of the digestion products of human immunoglobulin G (IgG) by papain. The results showed that papain immobilized on the monolithic rod through a spacer arm exhibits higher activity for the digestion of human IgG than that without a spacer arm. The apparent Michaelis-Menten kinetic constants of free and immobilized papain, K(m) and V(max), were determined. The digestion conditions of human IgG with free and immobilized papain were optimized. Comparison of the thermal stability of free and immobilized papain showed that the immobilized papain exhibited higher thermal stability than the free enzyme. The half-time of immobilized papain reaches about a week under optimum pH and temperature conditions.     

Preparation and properties of immobilized papain and lipase.

Papain and lipase were immobilized on derivatized Sepharose 4-B. The activated agarose had a binding capacity of 1.2 micronmol amino groups/ml packed agarose or 17 mg proteins/g dry agarose. The immobilized enzyme preparations were tested for the effects of pH of assay, temperature of assay, and substrate concentrations. The effect of 6M urea on the activity of papain was also determined. Soluble forms of the enzymes were used for comparison. Immobilization of the enzymes resulted in slightly different pH and temperature optima for activities. For immobilized papain Km(app) was similar to the one observed with soluble papain. Immobilization of lipase, however, cause a decrease in Km values. The immobilized enzyme preparations were stable when stored at 4 degrees C and pH 7.5 for periods up to eight months. The soluble enzymes lost their activity within 96 hr under similar storage conditions. Immobilized papain did not lose any activity after treatment with 6M urea for 270 min, whereas soluble papain lost 81% of its activity after the urea treatment, indicating that the immobilization of papain imparted structural and conformational stability to this enzyme.     

Cross-linking enzyme aggregates in the macropores of silica gel: A practical and efficient method for enzyme stabilization

Cross-linked enzyme aggregates of papain were prepared in commercial macroporous silica gel (CLEAs-MSG) in order to improve the operability and mechanical stability of CLEAs. CLEAs-MSG was obtained from simple adsorption, precipitation and one-step-cross-linking. CLEAs-MSG was characterized by stable structure that did not leak out enzyme from the macropores because of covalent bonding between CLEAs and MSG. The optimal temperature of papain CLEAs in MSG was 40–90 °C and the optimal pH was 7.0, which were improved compared to free papain and CLEAs. The CLEAs-MSG also enhanced the storage stability and thermal stability. Moreover, the CLEAs-MSG exhibited good reusability due to its suitable size and active properties. By using CLEAs-MSG of papain as biocatalyst, the kinetically controlled z-Ala-Gln synthesis was achieved with the yield of 32.9%, which was almost equal to that by using free papain as biocatalyst.     

An in vitro assessment of several enzymes for the supplementation of rabbit diets

In order to evaluate the effectiveness of several enzymes for use as feed supplements, the stabilities of these enzymes in relation to rabbit gastrointestinal content and feed processing conditions were investigated. Results showed that in glycine-HCl buffer pH 3.2, cellulase, papain and bromelain were acid tolerant while bacillus -amylase and protease were acid labile. When the buffer pH was 2.0, cellulase and papain still showed some pH resistance while bromelain was completely inactivated. With pepsin added to these buffer systems, similar results were obtained. In the gastric content system of pH 2.0, all the enzymes tested were unstable, but when the pH was 3.2 cellulase was fairly stable. Therefore, some factors in the gastric content other than pepsin might inactivate enzymes such as papain and bromelain. These factors might be the components of mineral premix in feed. On the other hand, preincubation of the enzymes with rabbit intestinal content would not affect the activities of these enzymes significantly. For the thermal stability study, it was found that, except for papain, thermal stabilities of all the enzymes tested could be significantly enhanced by mixing with the feed. Since mineral premix has an inhibitory effect on both bromelain and papain, the enzyme stabilization effect from feed could be caused by components other than mineral premix. Therefore, factors affecting the stabilities of each enzyme vary significantly and care must be taken when using these enzymes as feed supplements.     

Cysteine enhances activity and stability of immobilized papain

Immobilization of papain on Sepharose 6B in the presence of different concentrations of cysteine affected the enzyme activity depending on cysteine concentration. The maximum specific activity was observed when papain was immobilized with 200 mM cysteine. The immobilization process brought significant enhancement of stability to temperature and extreme pH values with respect to free papain. After immobilization, the optimum temperature of papain activity increased by 20°C (from 60 to 80°C) and its optimum pH activity shifted from 6.5 to 8.0. Catalytic efficiency (k_cat/K_m) and specific activity of the immobilized enzyme do not significantly change after immobilization. The temperature profile of this form of immobilized papain showed a broad range of activity compared with both free and immobilized form of papain in the absence of cysteine. This significant behavior in terms of activation energy is also discussed.     

Optimization of adsorption conditions of papain on dye affinity membrane using response surface methodology

The adsorption of papain on Reactive Blue 4 dye–ligand affinity membrane was investigated in a batch system. The combined effects of operating parameters such as initial pH, temperature, and initial papain concentration on the adsorption were analyzed using response surface methodology. The optimum adsorption conditions were determined as initial pH 7.05, temperature 39 °C, and initial papain concentration 11.0 mg/ml. At optimum conditions, the adsorption capacity of dye–ligand affinity membrane for papain was found to be 27.85 mg/g after 120 min adsorption. The papain was purified 34.6-fold in a single step determined by fast protein liquid chromatography. More than 85% of the adsorbed papain was desorbed using 1.0 M NaCl at pH 9.0 as the elution agent. The purification process showed that the dye–ligand immobilized composite membrane gave good separation of papain from aqueous solution.     

用木瓜蛋白酶及固定化木瓜蛋白酶拆分DL-苯丙氨酸

为了将手性化合物D-苯丙氨酸和L-苯丙氨酸进行分离,利用木瓜蛋白酶及固定化木瓜蛋白酶催化的方法对其拆分．试验结果表明,用DL-苯丙氨酸合成N-乙酰-DL-苯丙氨酸,得率为88.7％．木瓜蛋白酶、海藻酸钠 壳聚糖固定化木瓜蛋白酶(IPSAC)、尼龙布固定化木瓜蛋白酶(IPN)催化合成N-乙酰-L-苯丙氨酰苯胺时,对催化合成过程影响最大的因素分别是溶液中的离子强度、溶液中的离子强度、反应温度;溶液中的离子强度与pH对合成的影响较大．本试验得出分别用木瓜蛋白酶、IPSAC、IPN催化合成N-乙酰-L-苯丙氨酰苯胺的3个最佳方案;用此3个方案合成时,产率分别为61.2%、54.7%、36.3%．N-乙酰-L-苯丙氨酰苯胺水解生成L-苯丙氨酸,产率59.2%,光学纯度为96.6％．N-乙酰-D-苯丙氨酸水解生成D-苯丙氨酸,产率61.7%,光学纯度为95.7％．     

Beer-stabilizing and Antiedematic Activities of a New Chillproofing Enzyme

The colloidal stability of beer was examined after treatment with the chillproofing enzyme, “S-enzyme,” obtained from Serratia marcescens B–103. The beer-stabilizing activity of the S-enzyme was comparable to that of papain and the effects of the S-enzyme and papain were synergistic.

The antiedematic activity of the S-enzyme was examined in the hind paws of rats. When injected intraperitoneally, the antiedematic activity of the enzyme was approximately four times that of papain.     

Enzymatic hydrolysis and extraction of arachidonic acid rich lipids from Mortierella alpina

A novel method for efficient arachidonic acid rich lipids extraction was investigated. Six different enzymes (papain, pectinase, snailase, neutrase, alcalase and cellulase) were used to extract lipids from Mortierella alpina. The effects of enzyme concentration, temperature and hydrolysis time on oil recovery were evaluated using factorial experimental design and polynomial regression for each enzyme. Hydrolysis time is found to be the most important parameter for all enzymes. The ratios of enzyme mixtures were also studied. It showed that the mixtures of pectinase and papain (5:3, v/v), pectinase and alcalase (5:1, v/v) were better combined effects on oil yields. The effects of hydrolysis time and temperature were then analyzed by response surface methodology, and oil recoveries were satisfactory (104.6% for pectinase and papain and 101.3% for pectinase and alcalase). In the whole process, the lipid composition was not affected by the enzyme treatments according to fatty acid profile.     

Protease inhibitors from ripened and unripened bananas.

The proteolysis of casein by trypsin, chymotrypsin and papain was inhibited by ripened and unripened bontha, poovan, nendran, cavendish and rasthali bananas. The inhibition of trypsin, chymotrypsin and papain by different ripened banana cultivars was much more than that of unripened banana cultivars. The trypsin and chymotrypsin inhibitory activity of ripened poovan was heat stable, resistant to pronase and partly stable to trypsin but the trypsin and chymotrypsin inhibitory activity of unripened poovan was stable to heat and resistant to pronase only. The partial stability of trypsin inhibitory activity and instability of papain inhibitory activity of ripened poovan to alkaline pH suggests that the inhibitory factors of trypsin and papain were dissimilar. The probable role of unripened banana papain inhibitors in curing stomach ulcers and antinutritional role of ripened banana trypsin inhibitors is discussed.     

An enzymatic deconjugation method for the analysis of small molecule active drugs on antibody-drug conjugates

Antibody-drug conjugates (ADCs) are complex therapeutic agents that use the specific targeting properties of antibodies and the highly potent cytotoxicity of small molecule drugs to selectively eliminate tumor cells while limiting the toxicity to normal healthy tissues. Two critical quality attributes of ADCs are the purity and stability of the active small molecule drug linked to the ADC, but these are difficult to assess once the drug is conjugated to the antibody. In this study, we report a enzyme deconjugation approach to cleave small molecule drugs from ADCs, which allows the drugs to be subsequently characterized by reversed-phase high performance liquid chromatography. The model ADC we used in this study utilizes a valine-citrulline linker that is designed to be sensitive to endoproteases after internalization by tumor cells. We screened several proteases to determine the most effective enzyme. Among the 3 cysteine proteases evaluated, papain had the best efficiency in cleaving the small molecule drug from the model ADC. The deconjugation conditions were further optimized to achieve complete cleavage of the small molecule drug. This papain deconjugation approach demonstrated excellent specificity and precision. The purity and stability of the active drug on an ADC drug product was evaluated and the major degradation products of the active drug were identified. The papain deconjugation method was also applied to several other ADCs, with the results suggesting it could be applied generally to ADCs containing a valine-citrulline linker. Our results indicate that the papain deconjugation method is a powerful tool for characterizing the active small molecule drug conjugated to an ADC, and may be useful in ensuring the product quality, efficacy and the safety of ADCs.     

Improved stability of the carbon nanotubes-enzyme bioconjugates by biomimetic silicification

Nano-materials have been applied in many fields due to their excellent characteristics, such as the high surface area-to-volume ratio, excellent physicochemical properties and biological compatibility. In this study, multi-walled carbon nanotubes (MWCNTs) were utilized to prepare MWCNTs-papain bioconjugates and then realized the immobilization of papain. MWCNTs functionalized with carboxyl- and amine- groups on their surface were used as immobilization carriers. The immobilization of papain on the functionalized MWCNTs through physical absorption was examined. The conjugates were denoted as MWCNTs-papain bioconjugates. To improve the stability, the bioconjugates were further coated by silica through the biomimetic silicification process that induced by papain (denoted as silica-coated bioconjugates). The as-prepared MWCNTs-papain bioconjugates and the silica-coated bioconjugates were characterized by scanning electron microscopy (SEM) and Fourier transform infrared (FTIR) spectroscopy. The preliminary results showed that the bioconjugates could retain most of the initial activity of papain. Compared to free papain and MWCNTs-papain bioconjugates, the silica-coated bioconjugates exhibited significantly improved thermal, pH and recycling stability. Comparisons of the kinetic parameters between MWCNTs-papain bioconjugates and the silica-coated bioconjugates revealed that the K_m value of the immobilized papain experienced a slight increase after silica coating, which suggested that the silica coating did not significantly hinder papain's access to substrate or release of product.     

Climate change disequilibrium of boreal permafrost peatlands caused by local processes

Boreal forest and tundra are the biomes expected to experience the greatest warming during the course of the next century. The transient responses of boreal peatlands to climate change could be more complex than a simple large release of carbon and rapid migrations of vegetation and permafrost. Here we used alternative models to demonstrate that local processes typical of permafrost peatlands control carbon and vegetation dynamics in ways that strongly mediate effects of regional temperature gradients. Regional temperature affected stability and thaw rate. Thaw rate increased with mean annual temperature, and rates have accelerated within the last 50 yr. Local factors exerted a strong influence on stability, the levels of which were highest in three of the four temperature zones studied along the shaded south-southwest edges of collapse scars. The presence of Sphagnum fuscum cover increased stability. In all zones, survey points with S. fuscum showed more than twice the stability of points with feather moss, lichen, or no vegetation. In a direct model comparison between regional and local control, local factors were more important. Our results suggest that local processes mediate the effects of regional climate, and an accurate representation of ecosystem dynamics benefits from both local and regional processes.     

Bioconjugation of papain on superparamagnetic nanoparticles decorated with carboxymethylated chitosan

Functionalized Fe3O4 nanoparticles decorated with carboxymethylated chitosan were developed and used as a novel magnetic support for the covalent conjugation of papain, one of the most important industrial proteases. The analyses of transmission electron micrographs (TEM) and X-ray diffraction (XRD) showed that the size and structure of functionalized Fe3O4 nanoparticles had no significant changes after conjugation with papain. Magnetic measurement revealed that the resultant papain-conjugated nanoparticles were superparamagnetic with a saturation magnetization of 59.3 emu/g. Analyses of Fourier transform infrared (FTIR) spectroscopy and measurement of zeta potentials confirmed the conjugation of papain with the functionalized Fe3O4 nanoparticles. Compared with the native papain, the conjugated papain was found to exhibit enhanced enzyme activity, better tolerance to the variations of medium pH and temperature, and improved storage stability as well as good reusability. Considering that the magnetic separation technique possesses the advantages of rapidity, high efficiency, cost-effectiveness, and lack of negative effect on biological activity, such a bioconjugate system may hold potential applications in food, pharmaceutical, leather, cosmetic, and textile industries.     

Studies on quantum dots synthesized in aqueous solution for biological labeling via electrostatic interaction

3-Mercaptopropyl acid-stabilized CdTe nanoparticles synthesized in aqueous solution are effectively bound to a biomacromolecule, papain, via electrostatic interaction. The conjugation between the nanoparticles and the papain is demonstrated by UV-Vis absorption, photoluminescence spectroscopy, transmission electron microscopy, and fluorescence micrographs. The biological activity of papain is maintained after the conjugation. The effects of the quantity of papain and the size of nanoparticles on the fluorescence characteristics of the CdTe-papain bioconjugates were studied.     

Stability response of alpine meadow communities to temperature and precipitation changes on the Northern Tibetan Plateau

Biomass temporal stability plays a key role in maintaining sustainable ecosystem functions and services of grasslands, and climate change has exerted a profound impact on plant biomass. However, it remains unclear how the community biomass stability in alpine meadows responds to changes in some climate factors (e.g., temperature and precipitation). Long‐term field aboveground biomass monitoring was conducted in four alpine meadows (Haiyan [HY], Henan [HN], Gande [GD], and Qumalai [QML]) on the Qinghai‐Tibet Plateau. We found that climate factors and ecological factors together affected the community biomass stability and only the stability of HY had a significant decrease over the study period. The community biomass stability at each site was positively correlated with both the stability of the dominant functional group and functional groups asynchrony. The effect of dominant functional groups on community stability decreased with the increase of the effect of functional groups asynchrony on community stability and there may be a ‘trade‐off’ relationship between the effects of these two factors on community stability. Climatic factors directly or indirectly affect community biomass stability by influencing the stability of the dominant functional group or functional groups asynchrony. Air temperature and precipitation indirectly affected the community stability of HY and HN, but air temperature in the growing season and nongrowing season had direct negative and direct positive effects on the community stability of GD and QML, respectively. The underlying mechanisms varied between community composition and local climate conditions. Our findings highlighted the role of dominant functional group and functional groups asynchrony in maintaining community biomass stability in alpine meadows and we highlighted the importance of the environmental context when exploring the stability influence mechanism. Studies of community stability in alpine meadows along with different precipitation and temperature gradients are needed to improve our comprehensive understanding of the mechanisms controlling alpine meadow stability.     

Effect of polyethylene glycols on the function and structure of thiol proteases

Thiol proteases are industrially significant proteins with catalytic efficiency. The effect of low, medium and high molecular-weight poly (ethylene glycol) (PEG- 400, 6000 and 20000) on the stability of thiol proteases (papain, bromelain and chymopapain) has been studied by activity measurements using synthetic substrate. Structural studies performed on papain by far UV circular dichroism spectroscopic measurements indicate that there is loss in secondary structure of the protein in presence of increasing concentration of PEGs. Intrinsic fluorescence measurements lead us to conclude that tryptophan residues of protein encounter non-polar microenvironment in presence of PEG solvent while acrylamide quenching shows greater accessibility of tryptophan residues of papain in presence of PEGs. Extrinsic fluorescence measurements lead us to conclude that PEGs bind to the hydrophobic sites on the protein and thus destabilize it. Thermal denaturation studies show that melting temperature of papain is decreased in presence of PEGs. Possible mechanism of destabilization is discussed next. The results imply that caution must be exercised in the use of PEGs with thiol proteases or hydrophobic proteins in general, for different industrial applications, even at room temperature.     

Influence of metal ions on structure and catalytic activity of papain

Papain is an endoprotease belonging to cysteine protease family. The catalytic activity of papain in presence of two different metal ions namely zinc and cadmium has been investigated. Both the metal ions are potent inhibitors of the enzyme activity in a concentration dependent manner. The enzyme loses 50% of its activity at 2 x 10(-4) M of CdCl2 and 4 x 10(-4) M of ZnCl2. It is completely inactivated above 1 x 10(-3) M concentration of either ZnCl2 or CdCl2. Of the two metal ions zinc with a ki value of 5 x 10(-5) M is a more potent inhibitor than cadmium which has a ki value of 8 x 10(-5) M. Both the metal ions have higher affinity for active site than the substrate. At concentrations above 1 x 10(-2) M of metal ions the inhibition is not reversible. Calorimetric studies showed decreased thermal stability of papain upon binding of these metal ions. Far UV circular dichroic spectral data showed only small changes in the beta-structure content upon binding of these metal ions. These data are also supported by decrease in the apparent thermal transition temperature of papain by 5 degrees C upon binding of metal ions indicating destabilization of the papain molecule. The mechanism of both partial and complete inactivation of papain in presence of these two metal ions both at lower and higher concentration has been explained.     

玫瑰色微球菌A-04的红色素鉴定及稳定性分析

从石油污染的土壤和水样中筛选出一株玫瑰色微球菌A-04 Micrococcus roseus,对其所产红色色素进行了分离,并初步鉴定了色素种类,基于对影响A-04色素稳定性的单因素分析基础之上,采用3因素3水平响应面分析法,进一步对影响色素稳定性的主要因素进行了优化分析。结果表明A-04所产红色色素为类胡萝卜素,对该菌株的色素稳定性的单因素条件分析,色素对环境条件的耐受性较好,在80℃、pH5. 0～8. 0等条件下依然能长时间保持鲜红而不褪色。经响应面优化分析表明:温度、pH和溶剂是影响该色素稳定性的主要因素,温度与溶剂的交互作用对色素稳定性的影响也较为明显。pH5. 0～8. 0之间时,在80℃范围内,温度越低,同时溶剂的极性越大,越有利于维持色素的稳定性。本研究结果为该色素的实际开发和应用奠定了基础。