Hydrolysis of proteins by peptide hydrolases of Antarctic krill,Euphausia superba

• 1.1. The hydrolysis of casein by peptide hydrolases of Antarctic krill, E. superba, has been
• 2.2. The peptide hydrolases studied included trypsin-like enzymes, carboxypeptidase A-type of enzymes, carboxypeptidase B-type of enzymes, and an aminopeptidase isolated from Antarctic krill.
• 3.3. The trypsin-like enzymes seemed to play a decisive role in the degradation of casein, whereas the carboxypeptidase A, carboxypeptidase B and the aminopeptidase had limited effect when acting on casein alone. When combined with the trypsin-like enzymes, the exopeptidases effected enhanced release of amino acids from the protein.
• 4.4. Based on the pattern of amino acids relased from casein by a crude extract of krill, and by the isolated peptide hydrolases either alone or in combination, it is concluded that the purified peptide hydrolases examined comprise the major enzymes responsible for the autoproteolytic activity of krill at neutral- to weakly alkaline pH.

     

Hydrolysis of acrylonitrile by nitrile-converting bacterial cells immobilized on fibrous carbon adsorbents

Cells of the Pseudomonas fluorescens strain C2 containing nitrilase and Rhodococcus ruber strain gt1 with nitrile hydratase activity have been immobilized by the use of adsorption on fibrous carbon materials. It has been shown that the maximum adsorption value of Rhodococcus cells is higher than that in pseudomonades, reaching 21 mg of dry cells/1 g of the carrier vs. 6 mg, respectively. Cell adsorption, compared to cell suspension, gives a significant rise in nitrilase activity (by 7.4 times, using Ural TM-4 as the carrier) and in the stability of nitrile hydratase activity (5 reaction cycles without loss of activity, using Carbopon-B-active). Immobilized biocatalysts were also obtained by cell growth from Ps. fluorescens strain C2 and Rhodococcus ruber strain gt1 on fibrous carbon adsorbents. Biocatalyst productivity was higher for both strains when the carbonized material Ural TM-4 was used as the carrier.     

Binding of human liver hydrolases by immobilized lectins.

The binding of 22 human liver hydrolase activities by immobilized lectins of six different carbohydrate specificities, namely alpha-D-mannose (glucose), D-N-acetylglucosamine, D-N-acetylgalactosamine, L-fucose, alpha-D-galactose and beta-D-galactose, were examined. Differences in binding among these enzymes and within specific enzymes were observed. For example, the neutral forms of alpha-mannosidase and beta-xylosidase were bound by the Ulex europaeus lectin I (specific for L-fucose), whereas the acidic forms were not. Bandierea simplicifolia lectin (specific for alpha-galactose) bound 65% of beta-glucuronidase activity; recycling experiments demonstrated complete binding of the enzyme that had been eluted with the competitor D-galactose and no binding of the fraction that was not initially bound. These results suggested the presence of two forms of this enzyme. Similar data were obtained for acidic beta-galactosidase activity. These experiments may provide the basis for the expanded use of immobilized lectins for purification and characterization of hydrolases and other glycoproteins.     

Hydrolysis of lactose by immobilized microorganisms.

Cells of Lactobacillus bulgaricus, Escherichia coli, and Kluyveromyces (Saccharomyces) lactis immobilized in polyacrylamide gel beads retained 27 to 61% of the beta-galactosidase activity of intact cells. Optimum temperature and pH and thermostability of these microbial beta-galactosidases were negligibly affected by the immobilization. Km values of beta-galactosidase in immobilized cells of L. bulgaricus, E. coli, and K. lactis toward lactose were 4.2, 5.4, and 30 mM, respectively. Neither inhibition nor activation of beta-galactosidase in immobilized L. bulgaricus and E. coli appeared in the presence of galactose, but remarkable inhibition by galactose was detected in the case of the enzyme of immobilized K. lactis. Glucose inhibited noncompetitively the activity of three species of immobilized microbial cells. These kinetic properties were almost the same as those of free beta-galactosidase extracted from individual microorganisms. The activity of immobilized K. lactis was fairly stable during repeated runs, but those of E. coli and L. bulgaricus decreased gradually. These immobilized microbial cells, when introduced into skim milk, demonstrated high activity for converting lactose to monosaccharides. The flavor of skim milk was hardly affected by treatment with these immobilized cells, although the degree of sweetness was raised considerably.     

Hydrolysis of lactose by immobilized microorganisms.

Cells of Lactobacillus bulgaricus, Escherichia coli, and Kluyveromyces (Saccharomyces) lactis immobilized in polyacrylamide gel beads retained 27 to 61% of the beta-galactosidase activity of intact cells. Optimum temperature and pH and thermostability of these microbial beta-galactosidases were negligibly affected by the immobilization. Km values of beta-galactosidase in immobilized cells of L. bulgaricus, E. coli, and K. lactis toward lactose were 4.2, 5.4, and 30 mM, respectively. Neither inhibition nor activation of beta-galactosidase in immobilized L. bulgaricus and E. coli appeared in the presence of galactose, but remarkable inhibition by galactose was detected in the case of the enzyme of immobilized K. lactis. Glucose inhibited noncompetitively the activity of three species of immobilized microbial cells. These kinetic properties were almost the same as those of free beta-galactosidase extracted from individual microorganisms. The activity of immobilized K. lactis was fairly stable during repeated runs, but those of E. coli and L. bulgaricus decreased gradually. These immobilized microbial cells, when introduced into skim milk, demonstrated high activity for converting lactose to monosaccharides. The flavor of skim milk was hardly affected by treatment with these immobilized cells, although the degree of sweetness was raised considerably.     

Carbohydrate recognition and lysis by bacterial peptidoglycan hydrolases

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Variable responses of formyl peptide receptor haplotypes toward bacterial peptides

The chemoattractant neutrophil formyl peptide receptor (FPR) binds bacterial and mitochondrial N-formylated peptides, which allows the neutrophils to find the bacterial source and/or site of tissue damage. Certain inflammatory disorders may be due in part to an impaired innate immune system that does not respond to acute bacterial damage in a timely fashion. Because the human FPR is encoded by a large number of different haplotypes arising from ten single-nucleotide polymorphisms, we examined the possibility that some of these haplotypes are functionally distinct. We analyzed the response of three common FPR haplotypes to peptides from Escherichia coli, Mycobacterium avium ssp. paratuberculosis, and human mitochondria. All three haplotypes responded similarly to the E. coli and mitochondrial peptides, whereas one required a higher concentration of the M. avium peptide fMFEDAVAWF for receptor downregulation, receptor signaling, and chemotaxis. This raises the possibility of additional bacterial species differences in functional responses among FPR variants and establishes a precedent with potentially important implications for our innate immune response against bacterial infections. We also investigated whether certain FPR haplotypes are associated with rheumatoid arthritis (RA) by sequencing FPR1 from 148 Caucasian individuals. The results suggested that FPR haplotypes do not significantly contribute toward RA. George J. Saari, Deceased.     

Novel alternatives to antibiotics: bacteriophages, bacterial cell wall hydrolases, and antimicrobial peptides

Extensive research has been conducted on the development of three groups of naturally occurring antimicrobials as novel alternatives to antibiotics: bacteriophages (phages), bacterial cell wall hydrolases (BCWH), and antimicrobial peptides (AMP). Phage therapies are highly efficient, highly specific, and relatively cost-effective. However, precautions have to be taken in the selection of phage candidates for therapeutic applications as some phages may encode toxins and others may, when integrated into host bacterial genome and converted to prophages in a lysogenic cycle, lead to bacterial immunity and altered virulence. BCWH are divided into three groups: lysozymes, autolysins, and virolysins. Among them, virolysins are the most promising candidates as they are highly specific and have the capability to rapidly lyse antibiotic-resistant bacteria on a generally species-specific basis. Finally, AMP are a family of natural proteins produced by eukaryotic and prokaryotic organisms or encoded by phages. AMP are of vast diversity in term of size, structure, mode of action, and specificity and have a high potential for clinical therapeutic applications.     

Hydrolysis of oligoribonucleotides by alpha-helical basic peptides.

Poly(Leu-Lys-Lys-Leu) increases markedly the rate of hydrolysis of oligoribonucleotides. The polypeptide adopts and alpha-helical conformation in water in the presence of salt. Non-helical poly(Pro-Lys-Lys-Leu) is much less active. Ac-Leu-Lys-Lys-Leu-NHEt has no hydrolytic activity. Oligotetrapeptides Ac-(Leu-Lys-Lys-Leu)n-NHEt with increasing chain-length have been prepared by solid phase synthesis to evaluate the critical chain-length required for the hydrolytic activity. It is possible to correlate the activity to the propensity to form alpha-helices.     

Hydrolysis of soyabean by papain immobilized on wood by radiation polymerization

Summary Papain was immobilized on wood chips by radiation polymerization without substantial loss of enzyme activity. The immobilized papain was used to hydrolyse soyabean meal and found to be stable upto 6 cycles of operation. Maximum hydrolysis occurred with 15% (W/V) immobilized matrix.     

Hydrolysis of soluble starch by rotary disc of immobilized glucoamylase

Immobilized glucoamylase sheet was prepared using soluble collagen prepared from cow hide powder as the support material. The immobilized glucoamylase sheet was attached to the rotary disc and the rates of hydrolysis of maltose and soluble starch in the tank were measured. Qualitative discussions are made of the effect of stirring speed of immobilized enzyme disc on the overall reaction rate.     

Signal peptidases and signal peptide hydrolases

Signal peptidases, the endoproteases that remove the amino-terminal signal sequence from many secretory proteins, have been isolated from various sources. Seven signal peptidases have been purified, two fromE. coli, two from mammalian sources, and three from mitochondrial matrix. The mitochondrial enzymes are soluble and function as a heterogeneous dimer. The mammalian enzymes are isolated as a complex and share a common glycosylated subunit. The bacterial enzymes are isolated as monomers and show no sequence homology with each other or the mammalian enzymes. The membrane-bound enzymes seem to require a substrate containing a consensus sequence following the –3, –1 rule of von Heijne at the cleavage site; however, processing of the substrate is strongly influenced by the hydrophobic region of the signal peptide. The enzymes appear to recognize an unknown three-dimensional motif rather than a specific amino acid sequence around the cleavage site. The matrix mitochondrial enzymes are metallo-endopeptidases; however, the other signal peptidases may belong to a unique class of proteases as they are resistant to chelators and most protease inhibitors. There are no data concerning the substrate binding site of these enzymes. In vivo, the signal peptide is rapidly degraded. Three different enzymes inEscherichia coli that can degrade a signal peptidein vitro have been identified. The intact signal peptide is not accumulated in mutants lacking these enzymes, which suggests that these peptidases individually are not responsible for the degredation of an intact signal peptidein vivo. It is speculated that signal peptidases and signal peptide hydrolases are integral components of the secretory pathway and that inhibition of the terminal steps can block translocation.     

Hydrolysis of milk lactose by immobilized beta-galactosidase-hen egg white powder

Immobilized beta-galactosidase was obtained by crosslinking the enzyme with hen egg white using 2% glutaraldehyde. The gel obtained could be lyophilized to give a dry enzyme powder. The pH optimum of both the soluble and immobilized enzyme was found to be 6.8. The immobilized enzyme showed a higher K(m) for the substrates. The extent of enzyme inhibition by galactose was reduced upon immobilization. The stability towards inactivation by heat, urea, gamma irradiation, and protease treatment were enhanced. The bound enzyme as tested in a batch reactor could be used repeatedly for the hydrolysis of milk lactose. The possible application of this system for small-scale domestic use has been suggested.     

Hydrolysis of supersaturated naringin solutions by free and immobilized naringinase

Hydrolysis of concentrated naringin solutions was easily carried out with free enzymes taking advantage of the stability of supersaturated solutions. To use immobilized enzymes for the same purpose, a supersaturated solution of the substrate, coming from a reservoir at 80-90°C, was circulated through a reactor containing the catalyst at 40 or 50°C and sent again to the reservoir. The action of a-rhamnosidase was faster in supersaturated solution than in suspensions of naringin, and column clogging and other problems of handling solid substrate and products were avoided. At high concentration the reaction was inhibited by the product rhamnose.     

细菌SGNH家族水解酶的研究进展

SGNH水解酶家族是一类在四个保守序列区上具有严格保守催化残基Ser、Gly、Asn和His的水解酶,该家族水解酶广泛存在于真核生物和原核生物中。细菌来源的SGNH水解酶家族具有来源广泛、功能多样且催化机制独特等特点,在细菌致病性、碳源代谢和天然产物合成等方面发挥重要生物学功能,在医药、化工、生物燃料和环境修复等领域具有广泛的应用潜力。本文从氨基酸序列、蛋白结构特征、酶学催化机制、细菌生理功能及应用领域等方面对细菌来源的SGNH水解酶家族成员进行综述。