Influence of commercial proteases on the proteolysis of enzyme modified cheese : a thesis presented in partial fulfilment of the requirements for the degree of Master of Technology in Food Technology at Massey University, Palmerston North, New Zealand

The influence of four commercial proteases, Protease A, Protease B, Protease C and a two enzyme blend Protease DE, on proteolysis in an enzyme modified cheese (EMC) base has been investigated. Also, a series of preliminary experiments to determine the basic characteristics of the four enzyme preparations in buffer systems has been undertaken. Generally, the exopeptidase activity of the four enzyme preparations was more stable than the endopeptidase activity of the preparations. The highest enzyme activity for all preparations was given at pH 6.5 and Protease B was found to be sensitive to chelating agents. In addition, Protease B was found to contain at least two exopeptidases. Residual protease activities in EMC using a 55% moisture cheese base were found to be 0.005%, 0.009%, 0.007% and 0.004% (w/v) for Protease A, Protease B, Protease C and Protease DE, respectively, following inactivation by heating at 95°C heating for 30 minutes. Under the same incubation conditions (0.15% enzyme at 40°C for 24 h), Protease DE gave greater proteolysis than the three other enzymes and Protease B was the weakest protease. EMC digestion with a combination of proteases was different from that obtained with individual proteases. The combinations of Protease A/Protease C, Protease DE/Protease C, Protease B/Protease C and Protease DE/Protease A showed that the higher the proportion of the former protease in the combinations, the higher the amounts of total amino acids produced in the EMC. The combinations of Protease A/Protease B and Protease B/Protease DE gave greater amounts of total amino acids with the ratio of each enzyme close to 50:50 than with the individual enzymes. With respect to the molecular mass distribution of peptides in the various EMC digestions, Protease DE produced the greatest amount of peptides of 3 or fewer residues and Protease C gave the greatest amount of more medium sized peptides with 11-20 residues. Compared with Protease C, Protease A was more efficient in giving small peptides, while Protease B gave the lowest levels of medium and small peptides, but a high level of free amino acids. In sensory testing, Protease DE produced EMC with a strong pungent and astringent flavour, Protease C gave bitterness, Protease A gave a sweet flavour at a low concentration but bitter flavours with a high concentration and Protease B produced more savoury flavour without bitterness

PENGARUH KONSENTRASI NaCl TERHADAP AKTIVITAS SPESIFIK PROTEASE EKSTRASELULER DARI BAKTERI HALOFILIK HASIL ISOLASI BITTERN TAMBAK GARAM MADURA

Protease halofil dapat dimanfaatkan pada proses fermentasi makanan seperti pada pembuatan kecap ikan. Penelitian ini bertujuan untuk memperoleh bakteri halofilik dari isolat bittern tambak garam Madura dan mengisolasi protease halofil ekstraseluler serta menentukan pengaruh konsentrasi NaCl terhadap aktivitas spesifik protease halofil. Bakteri halofilik ditumbuhkan pada media HSB (Halophlie Synthetic Broth). Penentuan aktivitas protease dilakukan dengan menggunakan substrat azokasein dan kadar protein diukur dengan menggunakan metode Lowry. Berdasarkan penelitian diperoleh bakteri halofilik isolat bittern tambak garam Madura yang tumbuh optimal pada konsentrasi NaCl 4 %(b/v), dengan aktivitas spesifik protease halofil ekstraseluler tertinggi pada fraksi 4 (60-80 %) sebesar 58,537 Unit/mg protein. Adanya penambahan garam NaCl akan meningkatkan aktivitas protease halofil. Pada penelitian ini, aktivitas spesifik protease halofil meningkat menjadi 113,78 Unit/mg protein dengan konsentrasi optimal NaCl 0,750 M

Phenotypic hypersusceptibility to multiple protease inhibitors and low replicative capacity in patients who are chronically infected with human immunodeficiency virus type 1

Increased susceptibility to the protease inhibitors saquinavir and amprenavir has been observed in human immunodeficiency virus type 1 (HIV-1) with specific mutations in protease (V82T and N88S). Increased susceptibility to ritonavir has also been described in some viruses from antiretroviral agent-naïve patients with primary HIV-1 infection in association with combinations of amino acid changes at polymorphic sites in the protease. Many of the viruses displaying increased susceptibility to protease inhibitors also had low replication capacity. In this retrospective study, we analyze the drug susceptibility phenotype and the replication capacity of virus isolates obtained at the peaks of viremia during five consecutive structured treatment interruptions in 12 chronically HIV-1-infected patients. Ten out of 12 patients had at least one sample with protease inhibitor hypersusceptibility (change ≤0.4-fold) to one or more protease inhibitor. Hypersusceptibility to different protease inhibitors was observed at variable frequency, ranging from 38% to amprenavir to 11% to nelfinavir. Pairwise comparisons between susceptibilities for the protease inhibitors showed a consistent correlation among all pairs. There was also a significant relationship between susceptibility to protease inhibitors and replication capacity in all patients. Replication capacity remained stable over the course of repetitive cycles of structured treatment interruptions. We could find no association between in vitro replication capacity and in vivo plasma viral load doubling time and CD4(+) and CD8(+) T-cell counts at each treatment interruption. Several mutations were associated with hypersusceptibility to each protease inhibitor in a univariate analysis. This study extends the association between hypersusceptibility to protease inhibitors and low replication capacity to virus isolated from chronically infected patients and highlights the complexity of determining the genetic basis of this phenomenon. The potential clinical relevance of protease inhibitor hypersusceptibility and low replication capacity to virologic response to protease inhibitor-based therapies deserves to be investigated further

Structure of protease-cleaved escherichia coliα-2-macroglobulin reveals a putative mechanism of conformational activation for protease entrapment

Bacterial -2-macroglobulins have been suggested to function in defence as broad-spectrum inhibitors of host proteases that breach the outer membrane. Here, the X-ray structure of protease-cleaved Escherichia coli -2-macroglobulin is described, which reveals a putative mechanism of activation and conformational change essential for protease inhibition. In this competitive mechanism, protease cleavage of the bait-region domain results in the untethering of an intrinsically disordered region of this domain which disrupts native interdomain interactions that maintain E. coli -2-macroglobulin in the inactivated form. The resulting global conformational change results in entrapment of the protease and activation of the thioester bond that covalently links to the attacking protease. Owing to the similarity in structure and domain architecture of Escherichia coli -2-macroglobulin and human -2-macro­globulin, this protease-activation mechanism is likely to operate across the diverse members of this group

In Vitro Stability of Phytase from Recombinant Bacteria E. Coli BL21 (DE3) EAS1-AMP

The objective of the research was to inquire the Km, Vm, activity, intracellular phytase stability exposed to pH variation, temperature variation and protease (pepsin and pancreas) in vitro. The phytase was produced from recombinant bacteria E. coli BL21(DE3) EAS1-AMP using 1.5 mM IPTG as inducer. Intracellular enzyme was extracted via freeze shock and centrifugation. Pure enzyme was acquired through NI-NTA agarose column. The enzyme was then tested for Km, Vm, phytase activity and stability against pH, temperature and protease. Treatment levels for stability against protease were P0: without protease, P1: addition of pepsin, P2: addition of pepsin and pancreas, and the data were statistically analyzed using analysis of variance of one-way Completely Randomized Design. Crude intracellular phytase had Vm 6.39 υM/sec, Km 34.82 υM, and 277 units activity. Intracellular phytas was stable at pH 4–6 and 0–550 C. Protease level influenced the activity of intracellular phytase (P<0.05). Intracellular phytase was stable against pepsin but not pancreas. Keywords: Km, Vm, activity, intracellular phytase, pH, temperature, protease

Predictability of IL-28B-polymorphism on protease-inhibitor-based triple-therapy in chronic HCV-genotype-1 patients: A meta-analysis

AIM: To investigate the predictability of interleukin-28B single nucleotide polymorphism rs12979860 with respect to sustained virological response (SVR) in chronically hepatitis C virus (HCV) genotype-1 patients treated with a protease-inhibitor and pegylated interferon-$\alpha$ (Peg-INF-$\alpha$) based triple-therapy. METHODS: We searched PubMed, the Cochrane Library and Web of Knowledge for studies regarding the interleukin 28B (IL-28B)-genotype and protease-inhibitor based triple-therapy. Ten studies with 2707 patients were included into this meta-analysis. We used regression methods in order to investigate determinants of SVR. RESULTS: IL-28B-CC-genotype patients achieved higher SVR rates (odds 5.34, CI: 3.81-7.49) than IL-28B-non-CC-genotype patients (1.88, CI: 1.43-2.48) receiving triple-therapy. The line of therapy (treatment-na\"ive or -experienced for Peg-INF-$\alpha$) did not affect the predictive value of IL-28B (p=0.1). IL-28B-CC-genotype patients treated with protease inhibitor-based triple-therapy consisting of Boceprevir, Simeprevir, Telaprevir or Vaniprevir showed odds of 1.86, 9.77, 4.51 and 0.89, respectively. The odds for CC genotype patients treated with Faldaprevir cannot be quantified, as only a single study with a 100% SVR rate was available. CONCLUSION: IL-28B-SNP predicts the outcome for chronic HCV genotype-1 patients receiving protease inhibitor-based triple-therapy. The predictive value varies between the different protease inhibitors.Comment: 21 pages, 3 figures, 1 tabl

In silico prediction of mutant HIV-1 proteases cleaving a target sequence

HIV-1 protease represents an appealing system for directed enzyme re-design, since it has various different endogenous targets, a relatively simple structure and it is well studied. Recently Chaudhury and Gray (Structure (2009) 17: 1636 -- 1648) published a computational algorithm to discern the specificity determining residues of HIV-1 protease. In this paper we present two computational tools aimed at re-designing HIV-1 protease, derived from the algorithm of Chaudhuri and Gray. First, we present an energy-only based methodology to discriminate cleavable and non cleavable peptides for HIV-1 proteases, both wild type and mutant. Secondly, we show an algorithm we developed to predict mutant HIV-1 proteases capable of cleaving a new target substrate peptide, different from the natural targets of HIV-1 protease. The obtained in silico mutant enzymes were analyzed in terms of cleavability and specificity towards the target peptide using the energy-only methodology. We found two mutant proteases as best candidates for specificity and cleavability towards the target sequence

A neutrophil-dependent pathway for the generation of a neutral peptide mediator: partial characterization of components and control by alpha-1-antitrypsin.

A biologically active neutral peptide mediator is cleaved from a plasma protein substrate by an alpha-1-antitrypsin-inhibitable serine protease apparently residing on the membrane of the human neutrophil. The peptide mediator has an approximate mol wt of 1,000, and is distinguished from the kinin peptides by a neutral isoelectric point, susceptibility to inactivation by trypsin as well as chymotrypsin and activity on the isolated, atropinized, and antihistamine-treated guinea pig ileum with relatively little action on the estrous rat uterus. The neutrophil protease is fully inhibitable by DFP, trypsin inhibitors from lima or soy bean, and alpha-1-antitrypsin and is associated with the high mol wt fragments of the neutrophil and not the nuclear, lysosomal, or cytoplasmic subcellular fraction. The substrate has an approximate mol wt of 90,000 and is chromatographically separable from kininogen. The exquisite sensitivity of the neutrophil protease to alpha-1-antitrypsin was established both by inhibition with highly purified alpha-1-antitrypsin and by the inability of the protease to generate detectable neutral peptide in a homozygous (ZZ) alpha-1-antitrypsin-deficient patient without heat inactivation of the residual inhibitor. On the other hand, plasma from a (null) alpha-1-antitrypsin-deficient patient supported neutral peptide generation and revealed an additional factor which inactivated neutral peptide

Neural Network and Bioinformatic Methods for Predicting HIV-1 Protease Inhibitor Resistance

This article presents a new method for predicting viral resistance to seven protease inhibitors from the HIV-1 genotype, and for identifying the positions in the protease gene at which the specific nature of the mutation affects resistance. The neural network Analog ARTMAP predicts protease inhibitor resistance from viral genotypes. A feature selection method detects genetic positions that contribute to resistance both alone and through interactions with other positions. This method has identified positions 35, 37, 62, and 77, where traditional feature selection methods have not detected a contribution to resistance. At several positions in the protease gene, mutations confer differing degress of resistance, depending on the specific amino acid to which the sequence has mutated. To find these positions, an Amino Acid Space is introduced to represent genes in a vector space that captures the functional similarity between amino acid pairs. Feature selection identifies several new positions, including 36, 37, and 43, with amino acid-specific contributions to resistance. Analog ARTMAP networks applied to inputs that represent specific amino acids at these positions perform better than networks that use only mutation locations.Air Force Office of Scientific Research (F49620-01-1-0423); National Geospatial-Intelligence Agency (NMA 201-01-1-2016); National Science Foundation (SBE-0354378); Office of Naval Research (N00014-01-1-0624

Peptide synthesis by recombinant Fasciola hepatica cathepsin L1

Synthesis of the tripeptide Z-Phe-Arg-SerNH2 has been accomplished by a recombinant cysteine protease, cathepsin L1 from liver fluke (Fasciola hepatica), using Z-Phe-Arg-OMe as acyl acceptor and SerNH2 as nucleophile in 0.1 M ammonium acetate pH 9.0–12.5% v/v acetonitrile at 37 °C. LC–MS detection indicated tripeptide formation after 10 min, continuing up to 5.5 h. The ester Z-Phe-Arg-OMe was detected throughout the experiment but the hydrolysis product Z-Phe-Arg-OH appeared early and in quite large amounts. We believe that this is the first application of a parasite protease in enzymatic peptide synthesis