脉冲电场对木瓜蛋白酶影响的荧光光谱分析

木瓜蛋白酶溶液在电场强度为50 kV·cm-1,频率1 500 Hz,脉冲宽度40 μs的脉冲电场下接受19 800个脉冲处理后,其活性降低了56.5％。文章采用荧光偏光光谱对处理前后的样品进行了分析。 处理后酶样的荧光强度明显大于处理前,在峰值位置其荧光强度增加超过50％,峰位从342 nm移到了346 nm左右,其荧光偏振幅度明显减小。由此推断出木瓜蛋白酶经脉冲电场处理后酶蛋白的α-螺旋结构松散拉伸,分子内部的氨基酸残基暴露于分子表面,并有部分发生离解游离于溶液中,导致活性部位的结构发生变化,最终导致酶失活。     

Functional and bioactive properties of Larimichthys polyactis protein hydrolysates as influenced by plasma functionalized water-ultrasound hybrid treatments and enzyme types

The effects of plasma functionalized water (PFW) and its combination with ultrasound (UPFW) on the functional and bioactive properties of small yellow croaker protein hydrolysates (SYPHs) produced from three enzymes were investigated. Fluorescence and UV–Vis spectroscopy indicated that SYPHs tended to unfold with increasing intensity and shift in wavelengths to more flexible conformations under PFW and UPFW treatments. Particle size distribution and microstructure analysis revealed that treatments could disrupt aggregation of protein molecules to increase the roughness, specific surface area, and decrease the particle size of peptides during hydrolysis. The partially denatured structure of SYPHs induced by treatments increased the susceptibility of the fish proteins to exogenous enzymes, thereby accelerating the hydrolytic process to yield peptides with improved solubility, decreased emulsifying and foaming properties, and improved enzyme-specific antioxidant properties. The results revealed that the functionality of SYPHs was influenced by the treatment method and the enzyme type employed.     

Influence of sodium alginate pretreated by ultrasound on papain properties: Activity,structure, conformation and molecular weight and distribution

The aim of the study was to investigate the impact of sodium alginate (ALG) pretreated by ultrasound on the enzyme activity, structure, conformation and molecular weight and distribution of papain. ALG solutions were pretreated with ultrasound at varying power (0.05, 0.15, 0.25, 0.35, 0.45 W/cm²), 135 kHz, 50 °C for 20 min. The maximum relative activity of papain increased by 10.53% when mixed with ALG pretreated by ultrasound at 0.25 W/cm², compared with the untreated ALG. The influence of ultrasound pretreated ALG on the conformation and secondary structure of papain were assessed by fluorescence spectroscopy and circular dichroism spectroscopy. The fluorescence spectra revealed that ultrasound pretreated ALG increased the number of tryptophan on papain surface, especially at 0.25 W/cm². It indicated that ultrasound pretreatment induced molecular unfolding, causing the exposure of more hydrophobic groups and regions from inside to the outside of the papain molecules. Furthermore, ultrasound pretreated ALG resulted in minor changes in the secondary structure of the papain. The content of α-helix was slightly increased after ultrasound pretreatment and no significant change was observed at different ultrasound powers. ALG pretreated by ultrasound enhanced the stability of the secondary structure of papain, especially at 0.25 W/cm². The free sulfhydryl (SH) content of papain was slightly increased and then decreased with the increase of ultrasonic power. The maximum content of free SH was observed at 0.25 W/cm², under which the content of the free SH increased by 6.36% compared with the untreated ALG. Dynamic light scattering showed that the effect of ultrasound treatment was mainly the homogenization of the ALG particles in the mixed dispersion. The gel permeation chromatography coupled with the multi-angle laser light scattering photometer analysis showed that the molecular weight (M_w) of papain/ALG was decreased and then increased with the ultrasonic pretreatment. Results demonstrated that the activity of immobilized papain improved by ultrasonic pretreatment was mainly caused by the variation of the conformation of papain and the effect of interactions between papain and ALG. This study is important to explain the intermolecular interactions of biopolymers and the mechanism of enzyme immobilization treated by ultrasound in improving the enzymatic activity. As expected, ALG pretreated by appropriate ultrasound is promising as a bioactive compound carrier in the field of immobilized enzyme.     

Papain Catalyzed Oligomerization of α-Amino Acids. Synthesis and characterization of water-insoluble oligomers of L-methionine

Water-insoluble oligomers were synthesized from L -methionine ethyl ester with papain as the catalyst. L -Oligomethionine was obtained in yields of 50% when synthesis was carried out in highly concentrated citrate buffer at pH 5.5. Yields of up to 85% were obtained when the enzymatic synthesis proceeded in distilled water at pH 6.5, the pH being strictly maintained. The insoluble polymer was converted to highly water-soluble sulfoxide and sulfone derivatives, which consist mainly of an octamer with low amounts of heptamer or hexamer. Most of the carboxyl terminals still contained the ethyl ester group, only a minor part being present in the free acid form. The potential of the enzymatic approach for the synthesis of optically pure and monodisperse oligomers of α-amino acids is discussed.     

Radiation sterilization of enzyme hybrids with biodegradable polymers

Ionizing radiations, which have already been utilized for the sterilization of medical supplies as well as gas fumigation, should be the final candidate to decontaminate “hybrid” biomaterials containing bio-active materials including enzymes because irradiation induces neither heat nor substances affecting the quality of the materials and our health. In order to check the feasibility of ⁶⁰Co-gamma rays on these materials, we selected commercial proteases including papain and bromelain hybridized with commercial activated chitosan beads and demonstrated that these enzyme-hybrids suspended in water showed the significant radiation durability of more than twice as much as free enzyme solution at 25-kGy irradiation. Enhanced thermal and storage stability of the enzyme hybrids were not affected by the same dose level of irradiation, either, indicating that commercial irradiation sterilization method is applicable to enzyme hybrids without modification.     

An approach to computer-aided inhibitor design: Application to cathepsin L

Summary We have developed an approach to search for molecules that can be used as lead compounds in designing an inhibitor for a given proteolytic enzyme when the 3D structure of a homologous protein is known. This approach is based on taking the cast of the binding pocket of the protease and comparing its dimensions with that of the dimensions of small molecules. Herein the 3D structure of papain is used to model cathepsin L using the comparative modeling technique. The cast of the binding pocket is computed using the crystal structure of papain because the structures of papain and the model of cathepsin L are found to be similar at the binding site. The dimensions of the cast of the binding site of papain are used to screen for molecules from the Cambridge Structural Database (CSD) of small molecules. Twenty molecules out of the 80 000 small molecules in the CSD are found to have dimensions that are accommodated by the papain binding pocket. Visual comparison of the shapes of the cast and the 20 screened molecules resulted in identifying brevotoxin b, a toxin isolated from the red tide dinoflagellate Ptycho brevis (previously classified as Gymonodium breve), as the structure that best fits the binding pocket of papain. We tested the proteolytic activity of papain and cathepsin L in the presence of brevotoxin b and found inhibition of papain and cathepsin L with K_is of 25 M and 0.6 M, respectively. We also compare our method with a more elaborate method in the literature, by presenting our results on the computer search for inhibitors of the HIV-1 protease.     

Resonance Rayleigh scattering study on the interaction of gold nanoparticles with berberine hydrochloride and its analytical application

A heavy metals enzymatic-based assay using papain was developed. Papain was assayed using the Casein-coomassie-dye-binding assay. The assay is sensitive to several heavy metals. The IC₅₀ (concentration of toxicant giving 50% inhibition) of Hg²⁺, Ag²⁺, Pb², Zn²⁺ is 0.39, 0.40, 2.16, 2.11 mg l⁻¹, respectively. For Cu²⁺ and Cd²⁺ the LOQ (limits of quantitation) is 0.004 and 0.1 mg l⁻¹, respectively. The IC₅₀ and LOQ values were found to be generally comparable to several other enzymatic and bioassays tests such as: immobilized urease, 15-min Microtox™, 48 h Daphnia magna, and 96 h Rainbow trout. The papain assay is xenobiotics tolerant, has a wide pH for optimum activity, is temperature stable, and has a relatively quick assay time. The papain assay was used to identify polluted water samples from industrial sources in Penang, Malaysia. We found one site where the assay gave a positive toxic response. The toxicity of the site was confirmed using Atomic Emission Spectrometry analysis.     

STUDY ON THE IMMOBILIZATION OF PAPAIN WITH A MACROPOROUS BEAD CARRIER OF COPOLYMER CONTAINING MONOMER UNITS OF NAMINOETHYL ACRYLAMIDE AND VINYL ALCOHOI

A kind of macroporous bead carrier of copolymer containing monomer units of N-aminoethyl acrylamide and vinylalcohol was synthesized, i.e. the MR-AA carrier. Papain was immobilized on the carrier using glutaraldehyde as the couplingagent. The enzymatic activity of the immobilized papain was compared with free papain using casein as a substrate, and theeffects of glutaraldehyde concentration, pH, temperature, time and papain amount added on the activity recovery were alsoinvestigated. The results show that the MR-AA carrier contains reactive primary amine groups, hydrophilic amido links andhydroxyl groups, as well as macroporous structures based on its matrix (MR-AV matrix), furthermore, the activity recoveryof papain in the immobilization could reach 48%/～58%. In comparison with free papain, the resulting immobilized papainexhibits a remarkable thermostability and better reusability.     

Irradiation effects on hydrases for biomedical applications

To apply an irradiation technique to sterilize “Hybrid” biomedical materials including enzymes, we selected papain, a well-characterized plant endopeptidase as a model to examine durability of enzyme activity under the practical irradiation condition in which limited data were available for irradiation inactivation of enzymes. Dry powder and frozen aqueous solution of papain showed significant durability against ⁶⁰Co-gamma irradiation suggesting that, the commercial irradiation sterilizing method is applicable without modification. Although irradiation of unfrozen aqueous papain solution showed an unusual change of the enzymatic activity with the increasing doses, and was totally inactivated at 15 kGy, we managed to keep the residual activity more than 50% of initial activity after 30-kGy irradiation, taking such optimum conditions as increasing enzyme concentration from 10 to 100 mg/ml and purging with N₂ gas to suppress the formation of free radicals.     

Application of Enzymatic Promiscuity in Pharmaceutical Synthesis: Papain-catalyzed One-pot Synthesis of 1,4-Dihydropyridine Calcium Channel Antagonists and Derivatives

A new method for the synthesis of 1,4-dihydropyridine(1,4-DHP)calcium channel antagonists felodipme, nitrendipine and their derivatives via papain-catalyzed three-component reactions of aldehyde,methyl acetoacetate and ethyl 3-aminocrotonate was developed.Operational simplicity,mild reaction conditions and eco-friendliness are the key features of this protocol.