Toto Subroto

Chitinase and β-1,3-glucanase in the lutoid-body fraction of Hevea latex

Abstract The lutoid-body (bottom) fraction of latex from the rubber tree ( Hevea brasiliensis ) contains a limited number of major proteins. These are, besides the chitin-binding protein hevein, its precursor and the C-terminal fragment of this precursor, proteins with enzymic activities: three hevamine components, which are basic, vacuolar, chitinases with lysozyme activity, and a β-1,3-glucanase. Lutoid-body fractions from three rubber-tree clones differed in their contents of these enzyme proteins. The hevamine components and glucanase were isolated and several enzymic and structural properties were investigated. These enzymes are basic proteins and cause coagulation of the negatively charged rubber particles. The coagulation occurs in a rather narrow range of ratios of added protein to rubber particles, which indicates that charge neutralization is the determining factor. Differences in coagulation of rubber particles by lutoid-body fractions from various rubber clones can be explained by their content of hevamine and glucanase. Glucanase from the lutoid-body fraction may dissolve callus tissue and this may explain the observation that rubber-tree clones with a high glucanase content in this fraction produce more latex than clones with little glucanase. Sequence studies of two CNBr peptides of the glucanase indicate that this protein is homologous with glucanases from other plants, and that a C-terminal peptide, possibly involved in vacuolar targeting, may have been cleaved off.

Processed products of the hevein precursor in the latex of the rubber tree (Hevea brasiliensis)

The 20 kDa precursor of hevein and its C‐terminal 14 kDa domain have been isolated. Sequence analysis of the C‐terminal tryptic peptides of these proteins and comparison with the cDNA sequence indicate that they represent mature forms from which a C‐terminal propeptide, possibly involved in vacuolar targeting, has been removed. The molar ratio of hevein to the C‐terminal domain in the lutoid‐body fraction of rubber latex is about 30:1. This indicates that not only the pre‐ and propeptides but also the 14 kDa domain are removed by proteolysis or other processes in the latex vessel after the processing of hevein has taken place.

Proteolysis of α-amylase from Saccharomycopsis fibuligera: characterization of digestion products

Abstractα-Amylase from Saccharomycopsis fibuligera R-64 was successfully purified by butyl Toyopearl hydrophobic interaction chromatography, followed by Sephadex G-25 size exclusion and DEAE Toyopearl anion exchange chromatography. The enzyme has a molecular mass of 54 kDa, as judged by SDS PAGE analysis. Upon tryptic digestion, two major fragments with relative molecular masses of 39 kDa and 10 kDa, which resemble the A/B and C-terminal domains in the homologous Taka-amylase, were obtained and were successfully separated with the Sephadex G-50 size exclusion column. The 39-kDa fragment demonstrated a similar amylolytic activity to that of the undigested enzyme. However, it was found that the Km value of the 39-kDa fragment was about two-times higher than that of the undigested enzyme. Moreover, thermostability studies showed a lower half-life time for the 39-kDa fragment. These findings suggest that the 39-kDa fragment is the catalytic domain, while the 10-kDa fragment is the C-terminal one, which plays a role in thermostability and starch binding. Although the undigested enzyme is able to act on raw starches at room temperature, with maize starches as the best substrate, neither the undigested enzyme nor the fragments adsorb the tested raw starches. These results propose Saccharomycopsis fibuligera α-amylase as a raw starch-digesting but not adsorbing amylase, with a similar domain organization to that of Taka-amylase A.

Determination of cDNA and genomic DNA sequences of hevamine, a chitinase from the rubber tree Hevea brasiliensis

Hevamine is a chitinase from the rubber tree Hevea brasiliensis and belongs to the family 18 glycosyl hydrolases. This paper describes the cloning of hevamine DNA and cDNA sequences. Hevamine contains a signal peptide at the N-terminus and a putative vacuolar targeting sequence at the C-terminus which are not present in the mature protein. The gene encoding hevamine contains no introns. Southern hybridization of Hevea brasiliensis DNA with a full-length hevamine cDNA probe showed that there are multiple copies of genes encoding hevamine or hevamine homologues in the Hevea brasiliensis genome. In the lutoid body (vacuolar) fraction of rubber latex only three hevamine components were detected with very similar primary structures, which are probably products of one single locus on the genome.

Papaya (Carica papaya) lysozyme is a member of the family 19 (Basic, class II) chitinases

Abstract. The most comprehensive studies on a plant lysozyme (EC 3.2.1.17) are those on the enzyme from papaya (Carica papaya) latex, published in 1967 and 1969. However, the N-terminal amino acid sequence of five amino acid sequence of this enzyme, determined by manual Edman degradation, did not allow assignment to any of the much later-classified families of glycosyl hydrolases. N-Terminal sequence analysis of 22 residues of papaya lysozyme now shows unambiguously that the enzyme belongs to the family 19 chitinases. It has properties similar to those of basic class I chitinases with lysozyme activity, such as cleavage specificity at the C-1 of N-acetylmuramic acid with inversion of configuration, but as it lacks an N-terminal hevein domain, it should be classified as a class II chitinase.

The effluent of natural rubber factories is enriched in the antifungal protein hevein

Hevein is a small cystine-rich protein with a polypeptide chain length of 43 residues. It occurs in the lutoid body fraction of rubber latex, has affinity for chitin, and inhibits fungal growth. It was isolated from the effluent of a rubber factory in a yield of about 0.7 g/l. The elution position of the protein on reversed-phase HPLC, analysis by ion-spray mass spectrometry and its one-dimensional NMR spectrum indicated that it was identical to the protein isolated from the lutoid-body fraction. The total amount of other proteins in the effluent from the rubber production was less than 0.2 g/l. This means that the effluent of rubber factories is a very suitable source for the isolation of a protein that presumably has antifungal properties.

Effect of introducing a disulphide bond between the A and C domains on the activity and stability of Saccharomycopsis fibuligera R64 α-amylase.

Native enzyme and a mutant containing an extra disulphide bridge of recombinant Saccharomycopsis fibuligera R64 α-amylase, designated as Sfamy01 and Sfamy02, respectively, have successfully been overexpressed in the yeast Pichia pastoris KM71H. The purified α-amylase variants demonstrated starch hydrolysis resulting in a mixture of maltose, maltotriose, and glucose, similar to the wild type enzyme. Introduction of the disulphide bridge shifted the melting temperature (TM) from 54.5 to 56 °C and nearly tripled the enzyme half-life time at 65 °C. The two variants have similar kcat/KM values. Similarly, inhibition by acarbose was only slightly affected, with the IC50 of Sfamy02 for acarbose being 40 ± 3.4 μM, while that of Sfamy01 was 31 ± 3.9 μM. On the other hand, the IC50 of Sfamy02 for EDTA was 0.45 mM, nearly two times lower than that of Sfamy01 at 0.77 mM. These results show that the introduction of a disulphide bridge had little effect on the enzyme activity, but made the enzyme more susceptible to calcium ion extraction. Altogether, the new disulphide bridge improved the enzyme stability without affecting its activity, although minor changes in the active site environment cannot be excluded.

Computational Model of the Effect of a Surface-Binding Site on the Saccharomycopsis fibuligera R64 α-Amylase to the Substrate Adsorption

α-Amylase is one of the important enzymes in the starch-processing industry. However, starch processing requires high temperature, thus resulting in high cost. The high adsorptivity of α-amylase to the substrate allows this enzyme to digest the starch at a lower temperature. α-Amylase from Saccharomycopsis fibuligera R64 (Sfamy R64), a locally sourced enzyme from Indonesia, has a high amylolytic activity but low starch adsorptivity. The objective of this study was to design a computational model of Sfamy R64 with increased starch adsorptivity using bioinformatics method. The model structure of Sfamy R64 was compared with the positive control, ie, Aspergillus niger α-amylase. The structural comparison showed that Sfamy R64 lacks the surface-binding site (SBS). An SBS was introduced to the structure of Sfamy R64 by S383Y/S386W mutations. The dynamics and binding affinity of the SBS of mutant to the substrate were also improved and comparable with that of the positive control.

STABILIZATION OF VITAMIN A USING VITAMIN E AS ANTIOXIDANT IN LYOPHILIZED AUTOLOGOUS SERUM AND ITS ANTIBACTERIAL PROPERTIES

Dry eye syndrome is defined as an ocular surface disease caused by a lack of tear production or increased tear evaporation. Dry eye treatment with conventional drugs were less effective due to the nature and composition of the drugs that less akin to tears. Autologous serum is used as eyedrops in dry eye disease because it is natural and does not cause allergic reactions, as well as having biomechanical and biochemical properties that resemble normal tears. In liquid form, the content of serum didn’t last long, so the storage method of the serum was developed by using lyophilization with sucrose as lyoprotectant. The use of sucrose in lyophilization can only stabilize the protein content in serum so that the content of vitamin A in serum still decreased. In an effort to increase the stability of vitamin A in serum, this research was conducted to prepare freeze-dried autologous serum with the addition of vitamin E as an antioxidant of vitamin A. The purpose of this study was to determine the effect of vitamin E in stabilizing the concentration of vitamin A in freeze-dried autologous serum and checking its antibacterial properties by total plate count method. In this method, the separated blood serum was diluted with 0.9% sodium chloride followed by adding sucrose 60mM and vitamin E in different concentrations 0.5; 1; 2.5; 5% then this preparation was lyophilized. After that the serum was stored at 4°C and the content of vitamin A was analyzed every month by HPLC. Freeze-dried autologous serum with 1% addition of vitamin E showed the best results in stabilizing the vitamin A content up to 2 months. The content of vitamin A left in the 2-month freeze-dried serum was 0.0191-0.0761 ppm.

The Importance of Surface-Binding Site towards Starch-Adsorptivity Level in α-Amylase: A Review on Structural Point of View

Starch is a polymeric carbohydrate composed of glucose. As a source of energy, starch can be degraded by various amylolytic enzymes, including α-amylase. In a large-scale industry, starch processing cost is still expensive due to the requirement of high temperature during the gelatinization step. Therefore, α-amylase with raw starch digesting ability could decrease the energy cost by avoiding the high gelatinization temperature. It is known that the carbohydrate-binding module (CBM) and the surface-binding site (SBS) of α-amylase could facilitate the substrate binding to the enzymes active site to enhance the starch digestion. These sites are a noncatalytic module, which could interact with a lengthy substrate such as insoluble starch. The major interaction between these sites and the substrate is the CH/pi-stacking interaction with the glucose ring. Several mutation studies on the Halothermothrix orenii, SusG Bacteroides thetaiotamicron, Barley, Aspergillus niger, and Saccharomycopsis fibuligera α-amylases have revealed that the stacking interaction through the aromatic residues at the SBS is essential to the starch adsorption. In this review, the SBS in various α-amylases is also presented. Therefore, based on the structural point of view, SBS is suggested as an essential site in α-amylase to increase its catalytic activity, especially towards the insoluble starch.