Natural v/s Commercial Trypsin Isolated from Liver Waste of Labeo Rohita

The mounting human population and the massive amount of waste generated from the same is receiving particular attention towards valorization of waste. According to the annual report of FAO, (2018) the human consumption of fish protein has reached 87% in 2016 from 67% during 1960s. Aquaculture only has contributed to 5.8% annual growth rate among food sectors in the past decade. In this milieu, disposal of fish visceral waste is becoming a major menace to fishery industries exerting a great economic and environmental impact. Being perishable in nature, the organic portion of the waste decomposes rapidly and acts as a breeding ground for microbes. Moreover, the hefty and indiscriminate use of antibiotics and disinfectants in farmed animals is developing resistant strains, thus raising environment and ecological concerns. In order to solve such problem, the present investigation focussed upon employing the visceral trypsin as a cell dissociating agent. The efficacy of trypsin obtained from viscera of Labeo rohita upon KB cell line (Doubling time 50 hrs) was assessed in terms of cell viability. The cytotoxic effect of the visceral trypsin at 0.01%, 0.1% and 1% concentration were investigated at three time points (10 sec, 15 sec and 20 sec). Commercial (bovine) trypsin was considered as control. A time dependent decrease in cell viability upon gradually increasing the concentration was observed in all groups of treatment. The lowest reduction in cell viability (2%) was observed with 1% concentration at 15 sec and 20sec. Although, commercial trypsin was found more efficient than trypsin isolated from waste during this study but the potency of visceral trypsin observed cannot be ruled out. Thus, the application of this enzyme as a cell-dissociating agent suggested it as a comparable candidate with commercial trypsin.

Structural studies of complexes of kallikrein 4 with wild-type and mutated forms of the Kunitz-type inhibitor BbKI

Structures of BbKI, a recombinant Kunitz-type serine protease inhibitor from Bauhinia bauhinioides, complexed with human kallikrein 4 (KLK4) were determined at medium-to-high resolution in four crystal forms (space groups P3121, P6522, P21 and P61). Although the fold of the protein was virtually identical in all of the crystals, some significant differences were observed in the conformation of Arg64 of BbKI, the residue that occupies the S1 pocket in KLK4. Whereas this residue exhibited two orientations in the highest resolution structure (P3121), making either a canonical trypsin-like interaction with Asp189 of KLK4 or an alternate interaction, only a single alternate orientation was observed in the other three structures. A neighboring disulfide, Cys191–Cys220, was partially or fully broken in all KLK4 structures. Four variants of BbKI in which Arg64 was replaced by Met, Phe, Ala and Asp were expressed and crystallized, and their structures were determined in complex with KLK4. Structures of the Phe and Met variants complexed with bovine trypsin and of the Phe variant complexed with α-chymotrypsin were also determined. Although the inhibitory potency of these variant forms of BbKI was lowered by up to four orders of magnitude, only small changes were seen in the vicinity of the mutated residues. Therefore, a totality of subtle differences in KLK4–BbKI interactions within the fully extended interface in the structures of these variants might be responsible for the observed effect. Screening of the BbKI variants against a panel of serine proteases revealed an altered pattern of inhibitory specificity, which was shifted towards that of chymotrypsin-like proteases for the hydrophobic Phe and Met P1 substitutions. This work reports the first structures of plant Kunitz inhibitors with S1-family serine proteases other than trypsin, as well as new insights into the specificity of inhibition of medically relevant kallikreins.

Clustering of atomic displacement parameters in bovine trypsin reveals a distributed lattice of atoms with shared chemical properties

Low-frequency vibrations are crucial for protein structure and function, but only a few experimental techniques can shine light on them. The main challenge when addressing protein dynamics in the terahertz domain is the ubiquitous water that exhibit strong absorption. In this paper, we observe the protein atoms directly using X-ray crystallography in bovine trypsin at 100 K while irradiating the crystals with 0.5 THz radiation alternating on and off states. We observed that the anisotropy of atomic displacements increased upon terahertz irradiation. Atomic displacement similarities developed between chemically related atoms and between atoms of the catalytic machinery. This pattern likely arises from delocalized polar vibrational modes rather than delocalized elastic deformations or rigid-body displacements. The displacement correlation between these atoms were detected by a hierarchical clustering method, which can assist the analysis of other ultra-high resolution crystal structures. These experimental and analytical tools provide a detailed description of protein dynamics to complement the structural information from static diffraction experiments.

Identification of lumbrokinase by fibrin-plate method, fibrin-zymography and liquid chromatography mass spectrometry

Lumbrokinase, extracted from the cultured earthworm of Eisenia fetida, has been widely used as biochemical medicine in China to prevent or treat thrombosis. In the present study, the mechanism of lumbrokinase was investigated using the fibrin plate method. The results revealed that lumbrokinase contained both fibrinolytic and kinase components. The method of fibrin-zymography was used to show the existence and activity of lumbrokinase, and we proved that the fibrin-zymogram gel could be adopted as the identification method of earthworm. Subsequently, the components were identified by mass spectrometry. According to the results, fibrinolytic related components existed in the drug. These proteins were further compared with other serine proteins. The result showed that the identified proteins were similar to human trypsin and bovine trypsin. Besides, some also exhibited similar characteristics with human plasminogen activators. The mentioned results demonstrated that lumbrokinase products contained two major groups of protein components, suggesting two different functions.

CHARACTERIZATION OF MONOHEADED TRYPSIN INHIBITORS FROM THE SEEDS OF ABELMOSCHUS MOSCHATUS L.

Objective: The objective of the present study was to characterize the monoheaded trypsin inhibitors, Abelmoschus moschatus trypsin inhibitor-I (AMTI-I) and AMTI-II from the seeds of A. moschatus with respect to their specificity, mode of action, and active site residues.Methods: Standard methods were followed in determining inhibitory activities of monoheaded inhibitors. IC50 values and inhibitory constants (Ki) of AMTI-I and AMTI-II were determined. Studies on complex formation and chemical modification of inhibitors were performed.Results: AMTI-I and AMTI-II were found to be serpins, strongly active against trypsin, moderately active against porcine elastase, Staphylococcus aureus protease, and Aspergillus oryzae protease. AMTI-I and AMTI-II have shown non-competitive type of inhibition toward bovine trypsin with Ki values of inhibitors for trypsin found to be 0.25±0.02 nM and 0.22±0.06 nM, respectively. Complex studies revealed the formation of stable 1:1 complex of trypsin with both AMTI-I and AMTI-II. Chemical modification of the functional groups of the inhibitors by selective reagents indicated that arginine residues are essential for their trypsin inhibitory activities.Conclusion: Investigations on the specificity of protease inhibitors are important for understanding their physiological role, control mechanisms involved in the regulation of proteolysis in biological systems and mode of action.

A distributed lattice of aligned atoms exists in a protein structure: A hierarchical clustering study of displacement parameters in bovine trypsin

Low-frequency vibrations are crucial for protein structure and function, but only a few experimental techniques can shine light on them. The main challenge when addressing protein dynamics in the terahertz domain is the ubiquitous water that exhibit strong absorption. In this paper, we observe the protein atoms directly using X-ray crystallography in bovine trypsin at 100 K while irradiating the crystals with 0.5 THz radiation alternating on and off states. We observed that the anisotropy of atomic displacements increases upon terahertz irradiation. Atomic displacement similarities develop between chemically related atoms and between atoms of the catalytic machinery. This pattern likely arise from delocalized polar vibrational modes rather than delocalized elastic deformations or rigid-body displacements. This method can ultimately reveal how the alignment of chemically related atoms and the underlying polar vibrational dynamics make a protein structure stable.

Resistance of maize cultivars to Sitophilus zeamais (Coleoptera: Curculionidae)

ABSTRACT: Five Zea mays cultivars (BRS Caatingueiro, BRS Gorutuba, BRS Sertanejo, BRS Asa Branca and BR 106) were evaluated considering their effect on the nutrition of the maize weevil Sitophilus zeamais, by analysis of total protein in adult fed with these cultivars and for the presence of lectins and trypsin inhibitors in grains. In addition, free-choice and no-choice assays were performed to investigate the resistance of grains of the Z. mays cultivars to an attack by S. zeamais. The BR 106 cultivar showed the lowest susceptibility index, followed by BRS Caatingueiro, BRS Asa Branca, BRS Sertanejo and BRS Gorutuba. The number of emerged adults in the Z. mays cultivars ranged from 213.17 to 74.0, and the lowest number of insects was recorded for the BR 106 cultivar. The insects were able to feed on grains of all cultivars, but the BR 106 cultivar showed the least reduction in dried biomass. Lectins were detected in extracts from BR 106, BRS Asa Branca, BRS Sertanejo and BRS Gorutuba, and the highest activity was shown by BR 106. The lowest protein assimilation was detected in the insects from treatments with BRS Asa Branca. The extracts from all cultivars were able to inhibit the activity of bovine trypsin, but this effect was not related to the resistance degree of Z. mays cultivars. The results suggest the resistance of BR 160 to the attack of S. zeamais, as well as indicating that the presence of lectin in the grains is the cause of this resistance.