Influence of azide incorporation on binding affinity by small papain inhibitors

In order to develop affinity-based biosensor platforms, appropriate ligands with a functional handle for immobilization onto a biosensor surface are required. To this end, a library of papain inhibitors was designed and synthesized, containing different azide linkers for subsequent immobilization by ‘click’ chemistry, in this particular case by copper-free, strain-promoted azide–alkyne cycloaddition (SPAAC). Furthermore, a molecular docking study was performed to obtain a better insight as to at which position such azide handles could be tolerated without affecting binding affinity. Although the azide moiety is small, in some cases its introduction strongly influenced the binding affinity. For one class of inhibitors a swapped binding mode was proposed to explain the results. In addition, a specific site for linker introduction was identified, which did not significantly affect the binding affinity.     

Inhibition of Papaya latex papain by photosensitive inhibitors. 1-(4,5-dimethoxy-2-nitrophenyl)-2-nitroethene and 1,1-dicyano-2-(4,5-dimethoxy-2-nitrophenyl)-ethene

1-(4,5-Dimethoxy-2-nitrophenyl)-2-nitroethene (1) was shown to be an irreversible inhibitor of papain (EC 3.4.22.2), causing a complete inhibition (120 min preincubation, pH 8.0), assuming that it attached to Cys-25 at the active site of the enzyme (while a short preincubation time caused activation). Only partial inhibition of papain was achieved, however, with 1,1-dicyano-2-(4,5-dimethoxy-2-nitrophenyl)-ethene (2), a compound synthesized in this work, which is also an irreversible inhibitor of papain. Since both compounds 1 and 2, and in each case of the inhibited enzyme, were 2-nitrobenzyl derivatives, they and the modified enzyme were expected to be photosensitive. Indeed, irradiation of the inhibited enzyme in the presence of mercaptoethanol resulted in a full recovery of the enzyme activity following inactivation with compound 1 (similar to our previous finding with -galactosidase) and up to 67% recovery following inhibition with compound 2.     

Scenedesmus obliquus: Antioxidant and antiviral activity of proteins hydrolyzed by three enzymes

Purpose

To obtain protein hydrolysates from fresh water green algae Scenedesmus obliquus by three different enzymes and evaluate its antioxidant and antiviral activity.

纤维素固相化木瓜蛋白酶

 本文用叠氮法制备了纤维素固相化木瓜蛋白酶(简称CMCP)。与相应酶液水解酪蛋白的反应相比,它表现出较低的酶活性,较高的最适pH值和较高的稳定性。CMCP的比活回收率约为24％,最适pH值向碱性范围移动约为0.5个单位。CMCP经60℃热处理,持续3h活性无明显下降,在4℃下保存127天,活性只下降了40％左右。对这些参数,本文都根据CMCP的结构特点进行了分析。 CMCP柱还表现出明显的对啤酒的防浊能力。过柱的啤酒,氨基酸的含量大大增加。     

Characterization of an active hydrophilic erythrocyte membrane acetylcholinesterase obtained by limited proteolysis of the purified enzyme

Purified human erythrocyte membrane acetylcholinesterase was subjected to limited proteolysis with papain. This treatment generated a hydrophilic form of the enzyme as determined by charge-shift crossed immunoelectrophoresis and by binding to phenyl-Sepharose. The hydrophilic enzyme was stable and its activity was independent of the presence of amphiphiles. Electroimmunochemical analysis showed no antigenic difference between the two enzyme forms. Although the proteolytic treatment only brought about a small change in molecular weight, marked differences in the hydrodynamic properties were encountered. The Stokes radius decreased from 8.2 to 5.9 nm and the sedimentation coefficient increased from 6.3 to 7.0 S. The results are consistent with the view that a short hydrophobic peptide responsible for the amphipatic character of acetylcholinesterase is removed by the treatment with papain.     

Screening of plant peptidases for the synthesis of arginine-based surfactants

Partially purified preparations with proteolytic activity, obtained from South American native plants, were used as biocatalysts in condensation reactions of N-protected arginine alkyl ester derivatives with decylamine and dodecylamine in low-water content systems. The final products are cationic surfactants with potential application as emulsifiers and preservatives. Most of the proteolytic extracts were obtained from latex of species belonging to the Asclepiadaceae family (araujiain from Araujia hortorum, asclepain c from Asclepias curassavica and funastrain from Funastrum clausum). Hieronymain was obtained from unripe fruits of Bromelia hieronymi (Bromeliaceae). Plant proteases from commercial sources (papain and bromelain) were also tested as catalysts in the same reactions. Araujiain and funastrain furnished good reaction conversions (60–84%, with a ratio synthesis/hydrolysis of 2–5) similar to those obtained with commercial papain. Moreover, araujiain was the biocatalyst which rendered the best conversions (60%) for the synthesis of the two novel Bz-Arg-NH-dodecylamide (Bz-Arg-NHC₁₂) and Bz-Arg-NH-decylamide (Bz-Arg-NHC₁₀) derivatives. Moderate to poor conversions (10–50%, showing a ratio synthesis/hydrolysis of 0.5–1) were achieved with asclepain c, hieronymain and bromelain. The screening presented in this work revealed that, although these are structurally similar, their behavior for the synthesis of this kind of products differ among them.     

Enhanced activity,enantioselectivity and stability of papain in asymmetric hydrolysis of d,l-p-hydroxyphenylglycine methyl ester with ionic liquid

Papain-mediated asymmetric hydrolysis of D,L-p-hydroxyphenylglycine methyl ester (D,L-HPGME) was examined in the ionic liquid 1-butyl-3-methylimidazolium tetrafluoroborate (BMIM·BF₄) and different solvents. The activity of the enzyme varied widely with change in BMIM·BF₄ concentration, with 12.5% (v/v) being the optimum BMIM·BF₄ concentration for the reaction. Papain displayed much higher hydrolytic activity and enantioselectivity in phosphate buffer solution of 12.5% (v/v) BMIM·BF₄ (pH 7.0) than in other media examined. Comparative studies on the kinetics and activation energy (E_a) of this reaction performed in different media showed a higher V_max, a lower K_m and a lower E_a for the reaction taking place in phosphate buffer solution of 12.5% (v/v) BMIM·BF₄ than in other media tested. The stability of papain at 45°C was considerably enhanced in BMIM·BF₄ as compared with aqueous buffer, 2-propanol and acetonitrile. A half-life time of 169 h was observed with BMIM·BF₄ in the presence of substrate, which was 9.2–16.8-fold higher than those with the other solvents. These results suggested that BMIM·BF₄ is an excellent reaction medium for this reaction.     

汞离子对木瓜蛋白酶结构的影响及抑制机理的研究

通过荧光光谱、紫外光谱、傅里叶红外光谱(FTIR)和远紫外圆二色光谱(Far-UV CD)对重金属Hg²⁺作用下木瓜蛋白酶的结构变化进行了定性定量的表征,探索Hg²⁺对木瓜蛋白酶变性的抑制机理。结果表明:Hg²⁺是木瓜蛋白酶的强抑制剂,10^－4mol/L的HgCl₂可使木瓜蛋白酶丧失90%的活性;随着金属Hg²⁺浓度的增加,蛋白质所处的微环境和二级结构发生了明显的改变,(α螺旋＋β折叠)结构总量减少,无规则卷曲含量增多,酶的二级结构由规则有序转向无序。     

Esterification of N-benzyloxycarbonyldipeptides in ethanol-water with immobilized papain

The esterification of some N-benzyloxycarbonyl (Z)-dipeptides in ethanol-containing water was investigated using papain as a catalyst. The esterification took place in ethanol containing a samll amount of water (2% v/v, pH 9) with free papain at room temperature. The yield (after 24 h) of the ethyl ester was in the range of 25% to 50%. Any peptide bond cleavage of the substrates was not observed during esterification, indicating that the unfavorable amidase activity of papain was well depressed under these conditions. However, dipeptides having a D-amino amino acid (Z-valyl-D-alanine) or a bulky amino acid (Z-valylvaline) at the C-terminal position could not be esterified. It was found that the immobilization of papain on Amberlite XAD-8 increased the yield of the ester significantly as compared with free papain. In the esterification of Z-valylalanine using immobilized papain, the optimum water content, pH of an added buffer, and temperature were found to be 2% (v/v), 9, and 40 degrees C, respectively. The water content affected the yield of the product ester significantly.(c) 1993 John Wiley & Sons, Inc.     

Reaction Medium Selection for An Enzymatic Peptide Synthesis in An Aqueous-Organic Two-Phase System

Efficient development of enzymatic synthesis in two-phase systems is closely related with appropriate selection of the reaction medium (especially the solvent and phase ratio). A selection procedure based on the calculation of the theoretically allowable conversion and product concentration is presented and applied to a peptide synthesis using papain. For the synthesis of the dipeptide Boc-Gly-Phe-OMe, the operating conditions have been determined, and the two-phase system to be used has been successfully selected (with trichloroethylene being the best solvent).