Use of alkaline proteases for ultrafiltration membrane cleaning

Ability of alkaline proteases to clean ultrafiltration membranes fouled during milk processing was determined in two alkaline UF membrane cleaner, Alconox and Ultrasil. Optimum hydrolysis of milk proteins was observed with 5 g enzyme/l and showed a linear increase in water flux from 37.6 l m–2 h to 212.4 l m–2 h after 0.67 h. Enzyme performance was synergistically enhanced in combination with Alconox cleaner and exhibited 100% flux restoration.     

Recovery of a marker strain of Escherichia coli from ozonated water by membrane filtration.

Selective and nonselective growth media were evaluated at two incubation temperatures, 35 and 44.5 degrees C, for the recovery of a nalidixic acid-resistant marker strain of Escherichia coli ATCC 11775 by membrane filtration from ozonated 0.05 M phosphate buffer (pH 6.9). There were significantly fewer bacteria recovered with the standard m-FC agar when compared with the same growth medium prepared without bile salts and rosolic acid. This effect was particularly noticeable at the elevated incubation temperature of 44.5 degrees C. These findings are contrary to previous work which concluded that the standard American Public Health Association membrane filtration procedure is suitable for recovery of fecal coliform indicator bacteria from ozonated wastewater.     

Cytochemistry of membrane proteases

Summary Membrane proteases that are detectable by cytochemical means are the classified exopeptidases, aminopeptidases A and M (or N), -glutamyl transpeptidase (which also acts as transferase), dipeptidyl peptidase IV and the endopeptidase, enteropeptidase (also known as enterokinase). Not yet classified are the possible expeptidase, tripeptidyl peptidase and endopeptidases I (Ala-endopeptidase) and II (Arg-endopeptidase). All these membrane proteases can be investigated with either chromogenic or fluorogenic procedures using synthetic peptide substrates. The most useful substrates are 4-methoxy-2-naphthylamine amino acids and peptides for cytochemical localizations at the light and electron microscope levels, for cytophotometric quantification and the study of membrane protease isoenzymes after analytical isoelectric focusing. Amino acid or peptide derivatives of naphthylamine AS can be recommended for light microscopical localization and cytofluorometric quantification, and 7-amino-4-methylcoumarin and 7-amino-4-trifluoromethylcoumarin amino acids and peptides for the development of enzyme bands after isoelectric focusing. Cytochemistry reveals the heterogeneity in the distribution and species differences of membrane proteases in adult cells, tissues and organs and during development. It also reveals some common localizations, such as in small intestinal enterocytes and proximal tubule cells. The species and organ differences are substantiated and extended considerably by isoelectric focusing in combination with methods for the cytochemical detection of proteases. In addition, continuous cytophotometry or cytofluorometry (section and cultured cell biochemistry) allows the kinetic characteristics, initial reaction rates and maximum activities of all membrane proteases to be determined.The physiological functions of the endopeptidases and exopeptidases are still a matter of debate. However, from cytochemical inhibition studies with natural peptide substrates, e.g. peptide hormones, there is increasing evidence that the proteases detected with synthetic peptides play a decisive role in many physiological circumstances, e.g. in endocrine regulation mechanisms or the regulation of blood pressure. In this respect, capillary endothelium-linked surface membrane proteases may be especially important.     

Analysis of IgG glycopeptides by alkaline borate gel filtration chromatography

A protocol for the characterization of IgG glycopeptides is described. Central to this scheme is the novel application of an alkaline borate buffer to gel filtration chromatography. The use of this buffer significantly enhances the resolution of glycopeptides. Furthermore, it results in the separation of a unique size class of glycopeptides derived from IgG secreted by murine hybridomas. Although predominantly neutral, these glycopeptides differ both qualitatively and quantitatively by lectin affinity chromatography from the other glycopeptides which are presumably derived from the Fc portion of IgG.     

Physicochemical properties of alkaline serine proteases in alcohol

The alkaline proteases subtilisin Carlsberg and alcalase possess substantial enzymatic activity even when dissolved in ethanol. The crude enzymes were purified by gel filtration and the main fractions suspended in ethanol to give a translucent suspension. Both the supernatant and the resuspended precipitate after high-speed centrifugation were found to have enzymatic activities. The solubility of subtilisin Carlsberg in anhydrous ethanol was found to be 45.1g/ml and that of alcalase was 48.1g/ml by Coomassie blue dye-binding method using bovine serum albumin as a standard. In the presence of water, the solubility of both enzymes increased with water content. The stability of enzymes incubated in ethanol was assayed by their amidase and transesterase activities using Ala-Ala-Pro-Phe-pNA as substrate in phosphate buffer (pH8.2) and Moz-Leu-OBzl as substrate in anhydrous ethanol, respectively. The soluble enzymes have a half-life of about 36 hr and that of suspended enzymes about 50 hr in the amidase activity assay, whereas the same soluble enzymes have a half-life of about several hours and that of suspended enzymes 1 h by the transesterase activity assay. The stability of both enzymes decreased as water concentration increased. The diastereoselectivity of the enzyme-catalyzed hydrolysis of diastereo pairs of tetrapeptide esters,l-Ala-l-Ala-(d-orl-)Pro-l-Phe-OMe andl-Ala-l-Ala-(d-orl-)Ala-l-Phe-OMe, in phosphate is as high as that of the transesterification of these substrates in ethanol. It is concluded that active sites and selectivity of alkaline serine proteases in anhydrous alcohol are probably very similar to those in aqueous solution in spite of the fact that a lower reactivity is usually associated with the enzymes in nonaqueous solvents.     

Extracellular acid and alkaline proteases from Candida olea

Candida olea 148 secreted a single acid protease when cultured at acidic pH. In unbuffered medium, the culture pH eventually became alkaline and a single alkaline protease was produced. This was the only proteolytic enzyme produced when the organism was grown in buffered medium at alkaline pH. Both proteolytic enzymes were purified to homogeneity (as assessed by SDS-PAGE). The Mr of the acid protease was 30900, the isoelectric point 4.5; optimum activity against haemoglobin was at 42 degrees C and pH 3.3. This enzyme was inactivated at temperatures above 46 degrees C and was inhibited by either pepstatin and diazoacetyl-norleucine methyl ester but was insensitive to inhibition by either 1,2-epoxy-3-(p-nitrophenoxy)-propane or compounds known to inhibit serine, thiol or metallo proteases. The acid protease contained 11% carbohydrate. The alkaline protease had an Mr of 23400 and isoelectric point of 5.4. The activity of this enzyme using azocoll as substrate above 42 degrees C and was inhibited by phenylmethyl-sulphonyl fluoride and irreversible inactivated by EDTA. The enzyme was also partially inhibited by DTT but was insensitive to either pepstatin or p-chloromercuribenzoic acid.     

Microbial alkaline proteases: from a bioindustrial viewpoint

Alkaline proteases are of considerable interest in view of their activity and stability at alkaline pH. This review describes the proteases that can resist extreme alkaline environments produced by a wide range of alkalophilic microorganisms. Different isolation methods are discussed which enable the screening and selection of promising organisms for industrial production. Further, strain improvement using mutagenesis and/or recombinant DNA technology can be applied to augment the efficiency of the producer strain to a commercial status. The various nutritional and environmental parameters affecting the production of alkaline proteases are delineated. The purification and properties of these proteases is discussed, and the use of alkaline proteases in diverse industrial applications is highlighted.     

Identification of latent periodicity in domains of alkaline proteases

Internal repeats in protein sequences have wide-ranging implications for the structure and function of proteins. A keen analysis of the repeats in protein sequences may help us to better understand the structural organization of proteins and their evolutionary relations. In this paper, a mathematical method for searching for latent periodicity in protein sequences is developed. Using this method, we identified simple sequence repeats in the alkaline proteases and found that the sequences could show the same periodicity as their tertiary structures. This result may help us to reduce difficulties in the study of the relationship between sequences and their structures.     

Recovery of bovine lysozyme from transgenic sugarcane stalks: extraction, membrane filtration, and purification

Increased industrial use of sugarcane (Saccharum spp. hybrid) for food and bioenergy has led to considerable improvements in its genetic transformation, which allowed the development of not only pest- and herbicide-resistant lines but also lines expressing high-value bioproducts and biopolymers. However, the economic benefits of using inexpensive transgenic plant systems for the production of industrial proteins could be offset by high downstream processing costs. In this work, transgenic sugarcane expressing recombinant bovine lysozyme (BvLz) was used to evaluate the feasibility of extraction and fractionation of recombinant proteins expressed in sugarcane stalks. Three pH levels (4.5, 6.0 and 7.5) and three salt concentrations (0, 50, and 150 mM NaCl) were tested to determine BvLz and total protein extractability. Two extraction conditions were selected to prepare BvLz extracts for further processing by cross-flow filtration, a suitable method for concentration and conditioning of extracts for direct applications or prior to chromatography. Partial removal of native proteins was achieved using a 100 kDa membrane but 20–30 % of the extracted BvLz was lost. Concentration of clarified extracts using a 3 kDa membrane resulted in twofold purification and 65 % recovery of BvLz. Loading of concentrated sugarcane extract on hydrophobic interaction chromatography (HIC) resulted in 50 % BvLz purity and 69 % recovery of BvLz.     

Production in sea-water of thermostable alkaline proteases by a halotolerant strain of Bacillus licheniformis

A halotolerant strain of Bacillus licheniformis, previously isolated from marine sediments, produced high protease activity during the early stationary phase of growth. The use of sea-water in the fermentation medium enhanced the production of this activity of 150%. After a partial purification, three different proteolytic enzymes could be detected, which were alkaline serine proteases, exhibiting optimal activity at pH 9.0 and at 70°C. The proteases were activated by NaCl, with a two, three-fold increase in activity and were stable in presence of 0.7% NaBO3, 0.5% NaClO and 3% H2 O2. © Rapid Science Ltd. 1998     

Characterization of tumor cell membrane serine proteases by polyacrylamide gel electrophoresis

Studies in our laboratory have indicated that tumor cell membrane-bound proteases are responsible for the ability of tumor cells to lyse normal cells in vitro. In order to evaluate the tumor cell membrane enzymes, a purified tumor cell membrane preparation was prepared and the nonionic detergent Triton X-100 was used to extract active enzymes from the cell membranes. The solubilized membrane enzymes were then studied by Triton X-100 polyacrylamide gel electrophoresis under non-denaturing conditions. Using this technique the tumor cell membranes were shown to contain esterproteases that reacted with the substrates alpha-naphthyl acetate and naphthol-AS-aminocaproate. These esterproteases were inhibited by diisopropyl fluorphosphate and tosyl lysine chloromethyl ketone but not by tosylamide phenylethyl chloromethyl ketone, soybean trypsin inhibitor p-chloromercuribenzene sulfonic acid; N-ethylmaleimide choline iodide, alpha-1-anti-trypsin. NaF, epsilon-aminocaproic acid, ethylenediamine tetraacetic acid, or eserine. SBTI affinity chromatography of the tumor cell membrane extract revealed that some of the serine esterproteases bound to the SBTI column. The proteolytic activity of the tumor cell membrane extract and a fraction eluted from the SBTI affinity column was demonstrated using casein. We conclude that the tumor cell membranes contain previously undescribed serine proteases that are identifiable by their esterase activity and inhibitor profiles in polyacrylamide gels.     

膜过滤细胞直接计数新方法

介绍了两种膜过滤细胞直接计数新方法,即不透明滤膜的金相显微镜细胞直接计数法和透明滤膜的生物显微镜细胞直接计数法的基本原理、仪器设备、分析和计算方法,并且将此方法与荧光显微镜细菌计数、血球计数板等方法进行了比较。实际计数结果表明,两种膜过滤细胞直接计数法配合了独特的酸碱处理法以分散聚团细胞,用于非环境样品细胞浓度的测定具有快速准确简便的特点。此外,该方法基本不受细胞大小的影响,除了适用于体型较大的藻类、酵母菌的快速计数以外,还可以清晰地分辨诸如光合细菌这样体积较小的细菌。     

Bacterial alkaline proteases: molecular approaches and industrial applications

Proteolytic enzymes are ubiquitous in occurrence, being found in all living organisms, and are essential for cell growth and differentiation. The extracellular proteases are of commercial value and find multiple applications in various industrial sectors. Although there are many microbial sources available for producing proteases, only a few are recognized as commercial producers. A good number of bacterial alkaline proteases are commercially available, such as subtilisin Carlsberg, subtilisin BPN' and Savinase, with their major application as detergent enzymes. However, mutations have led to newer protease preparations with improved catalytic efficiency and better stability towards temperature, oxidizing agents and changing wash conditions. Many newer preparations, such as Durazym, Maxapem and Purafect, have been produced, using techniques of site-directed mutagenesis and/or random mutagenesis. Directed evolution has also paved the way to a great variety of subtilisin variants with better specificities and stability. Molecular imprinting through conditional lyophilization is coming up to match molecular approaches in protein engineering. There are many possibilities for modifying biocatalysts through molecular approaches. However, the search for microbial sources of novel alkaline proteases in natural diversity through the "metagenome" approach is targeting a hitherto undiscovered wealth of molecular diversity. This fascinating development will allow the biotechnological exploitation of uncultured microorganisms, which by far outnumber the species accessible by cultivation, regardless of the habitat. In this review, we discuss the types and sources of proteases, protease yield-improvement methods, the use of new methods for developing novel proteases and applications of alkaline proteases in industrial sectors, with an overview on the use of alkaline proteases in the detergent industry.     

Fermentation air filtration upgrading by use of membrane cartridge filters

The evolution of the use of improved, hydrophobic air filters in industrial plant germinators and fermentors is described, with the improved filters replacing Fiberglas media. The history and problems associated with use of Fiberglas media are described, as are the advantages of the hydrophobic filter.