Applied Biochemistry and Biotechnology (v.90, #2)

Cytochrome-c detection by Trevor Alleyne; Jerome Joseph; Valerie Sampson (97-105).
Following a myocardial infarction (MI) cells die or are damaged and their contents leak into the blood circulation, resulting in elevated serum levels of various enzymes, proteins, and organic molecules. Over the past few decades, it has become standard practice to employ the detection of these elevated substances as markers for the confirmation of MIs and to monitor MI patients’ response to treatment. Although it has previously been shown that cytochrome-c, a small respiratory protein, is among those elevated, the lack of a suitable detection system has prevented its routine use in the diagnosis of MIs. We present a preliminary study in which chemiluminescence was employed to detect elevated levels of cytochrome-c in the serum of MI patients. The technique, which is specific for c-type proteins, is approx 30 times more sensitive than the traditional Coomassie blue stain and can detect as little as 0.03 µg of protein. It also has potential for diagnostic use in other diseases that are characterized by mitochondrial damage.
Keywords: Cytochrome-c ; myocardial infarction; myocardial marker

One- and two-stage upflow anaerobic sludge-bed reactor pretreatment of winery wastewater at 4–10°C by Sergey V. Kalyuzhnyi; Marina A. Gladchenko; Vladimir I. Sklyar; Yekaterina S. Kizimenko; Sergey S. Shcherbakov (107-124).
The operating performance of a single and two (in series) laboratory upflow anaerobic sludge-bed (UASB) reactors (2.7-L working volume, recycle ratio varied from 1:1 to 1:18) treating diluted wine vinasse was investigated under psychrophilic conditions (4–10°C). For a single UASB reactor seeded with granular sludge, the average organic loading rates (OLRs) applied were 4.7, 3.7, and 1.7 g of chemical oxygen demand (COD)/(L·d) (hydraulic retention times [HRTs] were about 1 d) at 9–11, 6 to 7, and 4 to 5°C, respectively. The average total COD removal for preacidified vinasse wastewater was about 60% for all the temperature regimes tested. For two UASB reactors in series, the average total COD removal for treatment of non-preacidified wastewater exceeded 70% (the average OLRs for a whole system were 2.2, 1.8, and 1.3 g of COD/[L·d] under HRTs of 2 d at 10, 7, and 4°C, respectively). In situ determinations of kinetic sludge characteristics (apparent V m and K m ) revealed the existence of substantial mass transfer limitations for the soluble substrates inside the reactor sludge bed. Therefore, application of higher recycle ratios is essential for enhancement of UASB pretreatment under psychrophilic conditions. The produced anaerobic effluents were shown to be efficiently posttreated aerobically: final effluent COD concentrations were about 0.1 g/L. Successful operation of the UASB reactors at quite low temperatures (4–10°C) opens some perspectives for application of high-rate anaerobic pretreatment at ambient temperatures.
Keywords: Biogas; chemical oxygen demand removal; psychrophilic conditions; vinasse; upflow anaerobic sludge-bed reactor

Affinity purification of secreted alkaline phosphatase produced by baculovirus expression vector system by Fuming Zhang; Michael W. Wolff; David Williams; Katie Busch; Sybil C. Lang; David W. Murhammer; Robert J. Linhardt (125-136).
Human secreted alkaline phosphatase (SEAP) was produced in a stablytransformed Spodoptera frugiperda Sf-9 insect cell line (Sfb4GalT) following infection with a recombinant Autographa californica multiple nuclear polyhedrovirus containing the SEAP gene under control of the polyhedrin promoter. An affinity chromatographic column prepared by linking 4-aminobenzylphosphonic acid to histidyl-expoxy-Sepharose was used to isolate SEAP from the cell supernatant following removal of cells and virus and 10-fold concentration through ultrafiltration. We found that the binding of SEAP on the affinity matrix follows the Langmuir isotherm model. In addition, either recycling SEAP sample through the column for 24 h or loading high SEAP concentrations resulted in a high-purity product. Some nonspecific binding of protein on the matrix occurred when low concentrations of SEAP sample were loaded. Finally, we found that SEAP binding occurs rapidly, i.e., within 30 min of adding the SEAP sample to the affinity matrix.
Keywords: Affinity chromatography; human secreted alkaline phosphatase; baculovirus expression vector system; insect cells; recombinant protein; protein purification

Digestive gland protease pH optima and specific activities determined in Penaeus indicus with casein, azocasein, Azocoll, and Congo red fibrin as substrates were pH 7.7–9.2, 210–371 µmol of tyrosine/mg of homogenate protein/min; pH 7.8, 36; pH 6.0–7.0, 7; and pH 8.9–9.2, 7A Δ0.001 U/mg of homogenate protein/min, respectively. Activity in the shrimp was stable during frozen storage but relatively labile and very low (1.043 azocasein units) in the Norwegian lobster, Nephrops norvegicus. The high activity in shrimp is significant in aquaculture and may be a source of proteolytic enzymes for industrial use. The rapid deterioration after landing may be a consequence of the high and stable activity. The low activity in the lobster may present a problem in culture and requires a more critical choice of feed as well as further investigation. 4-(2-Aminoethyl)-benzenesulfonyl fluoride hydrochloride was a very convenient, fast-acting, and effective inhibitor of shrimp trypsin and chymotrypsin but did not completely inhibit general protease activity in shrimp and had a negligible effect on the lobster. A significant component of that activity may be from nonserine proteases (such as the exoproteases carboxypeptidase A and B and the leucine aminopeptidases), whose proportion relative to the serine proteases may be greater in the lobster.
Keywords: Shrimp midgut gland digestive proteases; enzyme pH-activity profiles; casein; sulfanilamide azocasein; Azocoll; Congo red fibrin; enzyme inhibitors; proteolytic enzymes; Penaeus indicus ; Nephrops norvegicus

Thermozymes and their applications by Marieke E. Bruins; Anja E. M. Janssen; Remko M. Boom (155-186).
Enzymes from thermophilic microorganisms, thermozymes, have unique characteristics such as temperature, chemical, and pH stability. They can be used in several industrial processes, in which they replace mesophilic enzymes or chemicals. Thermozymes are often used when the enzymatic process is compatible with existing (high-temperature) process conditions. The main advantages of performing processes at higher temperatures are reduced risk of microbial contamination, lower viscosity, improved transfer rates, and improved solubility of substrates. However, cofactors, substrates, or products might be unstable or other side reactions may occur. Recent developments show that thermophiles are a good source of novel catalysts that are of great industrial interest. Thermostable polymer-degrading enzymes such as amylases, pullulanases, xylanases, proteases, and cellulases are expected to play an important role in food, chemical, pharmaceutical, paper, pulp, and waste-treatment industries. Considerable research efforts have been made to better understand the stability of thermozymes. There are no major conformational differences with mesophilic enzymes, and a small number of extra salt bridges, hydrophobic interactions, or hydrogen bounds seem to confer the extra degree of stabilization. Currently, overexpression of thermozymes in standard Escherichia coli allows the production of much larger quantities of enzymes, which are easy to purify by heat treatment. With wider availability and lower cost, thermophilic enzymes will see more application in industry.
Keywords: Thermophilic; thermozyme; enzyme; hyperthermophilic; thermostability