Scouting Enzyme Behavior

Experimental exploration of enzymatic response in a multi-dimensional context faces the challenge of an explosive number of possible milieu conditions. We address this problem with an evolutionary search strategy that scouts the physiochemical milieu space for unanticipated enzyme behavior and rewards the discovery of experimental conditions that yield surprises

EKSTRAKSI ENZIM PAPAIN GETAH BUAH PEPAYA UNTUK PROSES \ud DEPROTEINASI PADA PEMBUATAN KITIN DARI KULIT UDANG WINDU \ud DAN APLIKASINYA SEBAGAI BAHAN PENJERNIH SARI BUAH SIRSAK

Papaya can produce enzyme that often called the enzyme papain. Usually, Enzyme is found in the resin or fruit that is still young. Papaya resin (papain) contains quite a variety of enzyme that is proteolotik (The protein). This enzyme functions as a protein remover (deproteinase) on the skin of the tiger shrimp. Enzyme will be catalyst ligamen of peptyda or protein to simple compound such as amino acids. Currently chitin and chitosan widely used in the processing industry the production process, ie as cleaner on theju ice. Goal of this research is 1) to determine the influence of extraction enzyme papain from papaya fruit \ud resin in the deproteinase process, 2) to determine the influence of the enzyme papain of papaya fruit resin in chitin extraction on making chitin-chitosan and 3) To know the influence of the addition chitosan as cleaner of the soursoap juice. This research was conducted in Laboraturium Technology of Agriculture Faculty of Agriculture, University of Muhammadiyah Malang in April 2009 until complete. \ud The method used in this research is to use the Complete Random Design (CRL), which consists of 2 phases. First Phase, the addition of papain enzyme treatment with 4-level (P0: without papain enzyme, P1: 1% papain enzyme, P2: papain enzyme 3% and P3: 5% papain enzyme) and second Phasetreatment in addition chitosan on soursoap juice with 3 levels (K0: without Chitosan, K1: chitosan control and K2: chitosan papain 5%), each do 3 times a retrial. First Phase combination treatment is the best Chitosan papain treatment 5% produce rendemen 9.22%, 10.48% of water content, protein content 0.67%; chitosan gray level that is 1.19%. For chitosan as cleaner in the soursoap juice show that the addition of chitosan can use as cleaner, marked with pH test, colour intensity, that indicates the result \ud is very significantly with the juice that did not experience additional of chitosan

Degradation of Chloroaromatics: Purification and Characterization of a Novel Type of Chlorocatechol 2,3-Dioxygenase of Pseudomonas putida GJ31

A purification procedure for a new kind of extradiol dioxygenase, termed chlorocatechol 2,3-dioxygenase, that converts 3-chlorocatechol productively was developed. Structural and kinetic properties of the enzyme, which is part of the degradative pathway used for growth of Pseudomonas putida GJ31 with chlorobenzene, were investigated. The enzyme has a subunit molecular mass of 33.4 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Estimation of the native Mr value under nondenaturating conditions by gel filtration gave a molecular mass of 135 ± 10 kDa, indicating a homotetrameric enzyme structure (4 × 33.4 kDa). The pI of the enzyme was estimated to be 7.1 ± 0.1. The N-terminal amino acid sequence (43 residues) of the enzyme was determined and exhibits 70 to 42% identity with other extradiol dioxygenases. Fe(II) seems to be a cofactor of the enzyme, as it is for other catechol 2,3-dioxygenases. In contrast to other extradiol dioxygenases, the enzyme exhibited great sensitivity to temperatures above 40°C. The reactivity of this enzyme toward various substituted catechols, especially 3-chlorocatechol, was different from that observed for other catechol 2,3-dioxygenases. Stoichiometric displacement of chloride occurred from 3-chlorocatechol, leading to the production of 2-hydroxymuconate.

The role of the enzyme in the succinate-enzyme-fumarate equilibrium

The following is an account of an investigation into the role of the enzyme in the succinate-enzyme-fumarate equilibrium. The method consisted in the comparison of the value of the free energy change in this reaction obtained from oxidation-reduction potentials, with that calculated from the entropies and other physicochemical properties of succinic acid and fumaric acid

PCR-RFLP Using BseDI Enzyme for Pork Authentication in Sausage and Nugget Products

A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) using BseDI restriction enzyme had been applied for identifying the presence of pork in processed meat (beef sausage and chicken nugget) including before and after frying. Pork sample in various levels (1%, 3%, 5%, 10%, and 25 %) was prepared in a mixture with beef and chicken meats and processed for sausage and nugget. The primers CYTb1 and CYTb2 were designed in the mitochondrial cytochrome b (cyt b) gene and PCR successfully amplified fragments of 359 bp. To distinguish existence of porcine species, the amplified PCR products of mitochondrial DNA were cut by BseDI restriction enzyme. The result showed pig mitochondrial DNA was cut into 131 and 228 bp fragments. The PCR-RFLP species identification assay yielded excellent results for identification of porcine species. It is a potentially reliable technique for pork detection in animal food processed products for Halal authentication

Kinetic Characterization and X-ray Structure of a Mutant of Haloalkane Dehalogenase with Higher Catalytic Activity and Modified Substrate Range

Conversion of halogenated aliphatics by haloalkane dehalogenase proceeds via the formation of a covalent alkyl-enzyme intermediate which is subsequently hydrolyzed by water. In the wild type enzyme, the slowest step for both 1,2-dichloroethane and 1,2-dibromoethane conversion is a unimolecular enzyme isomerization preceding rapid halide dissociation. Phenylalanine 172 is located in a helix-loop-helix structure that covers the active site cavity of the enzyme, interacts with the Clβ of 1,2-dichloroethane during catalysis, and could be involved in stabilization of this helix-loop-helix region of the cap domain of the enzyme. To obtain more information about the role of this residue in dehalogenase function, we performed a mutational analysis of position 172 and studied the kinetics and X-ray structure of the Phe172Trp enzyme. The Phe172Trp mutant had a 10-fold higher kcat/Km for 1-chlorohexane and a 2-fold higher kcat for 1,2-dibromoethane than the wild-type enzyme. The X-ray structure of the Phe172Trp enzyme showed a local conformational change in the helix-loop-helix region that covers the active site. This could explain the elevated activity for 1-chlorohexane of the Phe172Trp enzyme, since it allows this large substrate to bind more easily in the active site cavity. Pre-steady-state kinetic analysis showed that the increase in kcat found for 1,2-dibromoethane conversion could be attributed to an increase in the rate of an enzyme isomerization step that preceeds halide release. The observed conformational difference between the helix-loop-helix structures of the wild-type enzyme and the faster mutant suggests that the isomerization required for halide release could be a conformational change that takes place in this region of the cap domain of the dehalogenase. It is proposed that Phe172 is involved in stabilization of the helix-loop-helix structure that covers the active site of the enzyme and creates a rigid hydrophobic cavity for small apolar halogenated alkanes.

Partitioning of starter bacteria and added exogenous enzyme activities between curd and whey during Cheddar cheese manufacture

peer-reviewedPartitioning of starter bacteria and enzyme activities was investigated at different stages of Cheddar cheese manufacture using three exogenous commercial enzyme preparations added to milk or at salting. The enzyme preparations used were: Accelase AM317, Accelase AHC50, Accelerzyme CPG. Flow cytometric analysis indicated that AHC50 or AM317 consisted of permeabilised or dead cells and contained a range of enzyme activities. The CPG preparation contained only carboxypeptidase activity. Approximately 90% of starter bacteria cells partitioned with the curd at whey drainage. However, key enzyme activities partitioned with the bulk whey in the range of 22%–90%. An increased level of enzyme partitioning with the curd was observed for AHC50 which was added at salting, indicating that the mode of addition influenced partitioning. These findings suggest that further scope exists to optimise both bacterial and exogenous enzyme incorporation into cheese curd to accelerate ripening.Department of Agriculture, Food and the Marin

Covalently bound substrate at the regulatory site triggers allosteric enzyme activation

The mechanism by which the enzyme pyruvate decarboxylase from yeast is activated allosterically has been elucidated. A total of seven three-dimensional structures of the enzyme, of enzyme variants or of enzyme complexes form two yeast species (three of them reported here for the first time) provide detailed atomic resolution snapshots along the activation coordinate. The prime event is the covalent binding of the substrate pyruvate to the side chain of cysteine 221, thus forming a thiohemiketal. This reaction causes the shift of a neighbouring amino acid, which eventually leads to the rigidification of two otherwise flexible loops, where one of the loops provides two histidine residues necessary to complete the enzymatically competent active site architecture. The structural data are complemented and supported by kinetic investigations and binding studies and provide a consistent picture of the structural changes, which occur upon enzyme activation