Studies on Mold Dextranases

Nine strains capable of producing dextranase were isolated from soil. Among them, a strain belonging to the genus Aspergillus was chosen as the best producer of the enzyme. The mold produced greater amounts of dextranase than those found in some strains in the genus Penicillium, when grown aerobically at 28°C for 5 to 6 days in medium containing 1% dextran, 1% NaNO₃ or polypeptone, 0.2% yeast extracts, 0.4% K₂HPO₄ and small amounts of inorganic salts, pH 8.5. From the comparative taxonomic experiments, the mold used here was identified to be a strain belonging to Aspergillus carneus.     

Studies on Mold Protease

An acid protease of Rhizopus chinensis was purified by sequential chromatographies on columns of Duolite A-2, Sephadex G-100 and CM-cellulose, and crystallized from aqueous acetone solution. The preparation was shown to be monodisperse on column chromatography of ion-exchange sephadex and on ultracentrifugal analysis. The enzyme was most active at pH values between 2.9 and 3.3 and was stable over the range of pH 2.8 to 6.5. The protease was markedly inactivated by ferric ions and sodium lauryl sulfate, whereas it was affected by neither sulfhydryl reagents nor metal-chelating agents. In milk-clotting activity, the acid protease was shown to be one of the most potent enzymes among those of fungal origin. Substrate specificity experiments on several synthetic peptides indicated that the peptide bonds susceptible to the action of the enzyme were mainly those involving amino group of aromatic amino acids.     

Studies on the Chitinolytie Enzymes of Black-koji Mold

In an attempt to separate the enzyme system participating in the decomposition of glycol chitin to constituent aminosugar, the purification of chitinase of Aspergillus niger was carried out by detemining both liquefying and saccharifying activities. Using fractionation with ammonium sulfate and column chromatography by hydroxylapatite, the chitinase system of the mold was separated into different enzyme fractions, which were required for the complete hydrolysis of glycol chitin. It was found that one of these enzymes caused a rapid decrease in viscosity of glycol chitin solution, another enzyme possessed N-acetyl-β-glucosaminidase activity upon N, N′-diacetylchitobiose and β-methyl-N-acetylglucosaminide, and that glycol chitin was decomposed to constituent aminosugar by a successive action of the two different enzymes.     

Studies on the Chitinolytic Enzymes of Black-koji Mold

The measurement of N-acetyl-β-glucosaminidase activity upon β-MAGA was carried out in order to survey oligosaccharidase fraction in the chitinolytic enzymes of Aspergillus niger. N-Acetyl-β-glucosaminidase was purified in parallel with chitobiase activity, being separated from chitinase activity, and some properties of the enzyme in the hydrolysis of β-MAGA and DACE were investigated. The enzyme hydrolysed more rapidly S-glueosaminidie bonds in DACE than that in β-MAGA, but did not decompose α-MAGA.     

Studies on the Mechanism of Mold Protease Production

The activating factors of the inactive protease in cell-free extracts obtained from growing mycelia of Aspergillus sojae KS were studied. It was found that the several kinds of metals were involved in activation, and the role of these metals on the activation was investigated. The velocity of the activation was maximal around pH 10 as well as around pH 5. It was proved that a kinase (enzyme) capable of activating the inactive protease in alkaline solution does exist in the cell-free extract.     

Studies on the Chitinolytic Enzymes of Black-koji Mold

The mode of degradation of glycol chitin and chitin by two enzyme fractions separated from Aspergillus niger was investigated. One of the enzyme rapidly cleaved the endo-β-glucosaminidic bonds in the polysaccharide chain, forming chitodextrin and oligosaccharides, while the other produced monosaccharide as a main product in the degradation. The successive action of the two enzymes was also examined. Intermediate products in the enzymatic degradation were surveyed using paper and column chromatography. Also, the over-all pattern of degradation of glycol chitin and chitin by the chitinase system of Aspergillus niger was discussed.     

Studies on the Chitinolytic Enzymes of Black-koji Mold

It has been found that glycol chitin is a suitable substrate for the viscometric determination of chitinase activity, because the viscosity of its aqueous solution is not affected by the presence of added salt and the changes of pH, differing from chitosan acetate and carboxy-methyl chitin used by earlier workers. Using this substrate the viscometric activity is determined, basing on the observation that the time required to halve the viscosity of reaction mixture is inversely proportional to the amount of enzyme used.     

Studies on the Chitinolytic Enzymes of Black-koji Mold

It was found that the purified chitinase preparation acts upon glycol chitin resulting in the decomposition to constituent aminosugar, the saccharifying activity being determined by application of the Morgan-Elson reaction. The enzymatic properties of the mold chitinase were investigated by measuring liquefying activity and saccharifying activity. Distinct differences were observed between the two activities, and especially liquefying activity was more stable than saccharifying activity against heat treatment. The chitinase preparation whose saccharifying activity was inactivated by heating was able to decrease the viscosity of glycol chitin solution, with an insignificant production of aminosugar.     

Studies on the Mechanism of Mold Protease Production

Inactive protease in the cell free extracts obtained from growing cells of Aspergillus sojae KS was collected in a supernatant of ultracentrifugation at 14×l0⁴ g, and in fractions obtained by acetone of 35~50 per cent and by ammonium sulfate of 0.5~0.6 saturation.

The inactive protease has the same resistance against pH or heat treatments as active protease has. The activation of the inactive protease was maximal between pH 5~6, and was accelerated by several kinds of protease, and was not affected by thioglycollate and KCN.     

曲酸生产菌的诱变选育

Aspergillusoryzae2336经两次紫外诱变后筛选出突变菌株UV21012-1。该菌株曲酸产量比出发菌株提高了1．68倍;遗传性状稳定,传代5次曲酸产量基本不下降,而且发酵周期也比出发菌株明显缩短。     

Studies on plant gums. Proteases in neem (Azadirachta indica) gum

Proteolytic activity was detected in neem (Azadirachta indica) exudate gum when tested with casein and albumin as substrates. The enzyme activity was separated into two fractions by chromatography on TEAE-cellulose after EDTA treatment. Both the enzyme fractions were fairly stable to high temperatures and wide range of pH conditions. The pH optima were found to be around 6.5. Phenylmethyl sulphonylfluoride inhibited the activity of both the fractions. EDTA, Β-mercaptoethanol, tosylamide phenylethylchloromethylketone, tosyllysine chloroimethylketone,p-chloromercuribenzoate and dithiobis-2-nitrobenzoie acid did not affect the activity of the two enzyme fractions. The two fractions had no hydrolytic action on a variety of synthetic substrates tested.     

Isolation of Alkaline and Neutral Proteases from Aspergillus flavus var. columnaris, a Soy Sauce Koji Mold

Two different extracellular proteases, protease I (P-I), an alkaline protease, and protease II (P-II) a neutral protease, from Aspergillus flavus var. columnaris were partially purified by using (NH(4))(2)SO(4) precipitation, diethylaminoethyl-Sephadex A-50 chromatography, carboxymethylcellulose CM-52 chromatography, and Sephadex G-100 gel filtration. The degree of purity was followed using polyacrylamide gel electrophoresis. The activity of P-I was completely inhibited by 0.1 mM phenylmethylsulfonyl fluoride, and that of P-II was completely inhibited by 1 mM ethylenediaminetetraacetate. By using these inhibitors with extracts of wheat bran koji, the proportions of total activity that could be assigned to P-I and P-II were 80 and 20%, respectively. This compared favorably with activities estimated by using polyacrylamide gel electrophoresis slices (82 and 18%, respectively). Extracts from factory-run soybean koji gave comparable results. Both enzymes demonstrated maximum activity at 50 to 55 degrees C and only small changes in activity between pH 6 and 11. For P-I, activity was somewhat higher from pH 8.0 to 11.0, whereas for P-II it was somewhat higher from pH 6 to 9. In the presence of 18% NaCl, the activities of both P-I and P-II dropped by approximately 90 and 85%, respectively. P-I was inferred to possess aminopeptidase activity since it could hydrolyze l-leucyl-p-nitroanilide hydrochloride. P-II was devoid of such activity. The ramifications of the results for factory-produced soy sauce koji are discussed.     

Chloroplast Proteases

The chloroplast within the plant cell has a dynamic environment where proteases play an important role in processing of precursor proteins, degradation of incomplete proteins lacking cofactors, stress-induced degradation and removal of damaged proteins. A number of proteases in the chloroplast are well characterized and found to be localized within different compartments such as stroma, thylakoids and lumen. In recent years, an increasing number of proteases in chloroplasts have been discovered and identified as bacterial protease homologues. These include the stromal Clp, thylakoidal FtsH and lumenal DegP. The current focus is to understand their role in chloroplast regulation both at the enzyme-substrate and genetic levels.     

Proteases in autophagy

Autophagy is a process involved in the proteolytic degradation of cellular macromolecules in lysosomes, which requires the activity of proteases, enzymes that hydrolyse peptide bonds and play a critical role in the initiation and execution of autophagy. Importantly, proteases also inhibit autophagy in certain cases. The initial steps of macroautophagy depend on the proteolytic processing of a particular protein, Atg8, by a cysteine protease, Atg4. This processing step is essential for conjugation of Atg8 with phosphatidylethanolamine and, subsequently, autophagosome formation. Lysosomal hydrolases, known as cathepsins, can be divided into several groups based on the catalitic residue in the active site, namely, cysteine, serine and aspartic cathepsins, which catalyse the cleavage of peptide bonds of autophagy substrates and, together with other factors, dispose of the autophagic flux. Whilst most cathepsins degrade autophagosomal content, some, such as cathepsin L, also degrade lysosomal membrane components, GABARAP-II and LC3-II. In contrast, cathepsin A, a serine protease, is involved in inhibition of chaperon-mediated autophagy through proteolytic processing of LAMP-2A. In addition, other families of calcium-dependent non-lysosomal cysteine proteases, such as calpains, and cysteine aspartate-specific proteases, such as caspases, may cleave autophagy-related proteins, negatively influencing the execution of autophagic processes. Here we discuss the current state of knowledge concerning protein degradation by autophagy and outline the role of proteases in autophagic processes. This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome.     

Proteases in autophagy

Autophagy is a process involved in the proteolytic degradation of cellular macromolecules in lysosomes, which requires the activity of proteases, enzymes that hydrolyse peptide bonds and play a critical role in the initiation and execution of autophagy. Importantly, proteases also inhibit autophagy in certain cases. The initial steps of macroautophagy depend on the proteolytic processing of a particular protein, Atg8, by a cysteine protease, Atg4. This processing step is essential for conjugation of Atg8 with phosphatidylethanolamine and, subsequently, autophagosome formation. Lysosomal hydrolases, known as cathepsins, can be divided into several groups based on the catalitic residue in the active site, namely, cysteine, serine and aspartic cathepsins, which catalyse the cleavage of peptide bonds of autophagy substrates and, together with other factors, dispose of the autophagic flux. Whilst most cathepsins degrade autophagosomal content, some, such as cathepsin L, also degrade lysosomal membrane components, GABARAP-II and LC3-II. In contrast, cathepsin A, a serine protease, is involved in inhibition of chaperon-mediated autophagy through proteolytic processing of LAMP-2A. In addition, other families of calcium-dependent non-lysosomal cysteine proteases, such as calpains, and cysteine aspartate-specific proteases, such as caspases, may cleave autophagy-related proteins, negatively influencing the execution of autophagic processes. Here we discuss the current state of knowledge concerning protein degradation by autophagy and outline the role of proteases in autophagic processes. This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome.     

Proteases as therapeutics

Proteases are an expanding class of drugs that hold great promise. The U.S. FDA (Food and Drug Administration) has approved 12 protease therapies, and a number of next generation or completely new proteases are in clinical development. Although they are a well-recognized class of targets for inhibitors, proteases themselves have not typically been considered as a drug class despite their application in the clinic over the last several decades; initially as plasma fractions and later as purified products. Although the predominant use of proteases has been in treating cardiovascular disease, they are also emerging as useful agents in the treatment of sepsis, digestive disorders, inflammation, cystic fibrosis, retinal disorders, psoriasis and other diseases. In the present review, we outline the history of proteases as therapeutics, provide an overview of their current clinical application, and describe several approaches to improve and expand their clinical application. Undoubtedly, our ability to harness proteolysis for disease treatment will increase with our understanding of protease biology and the molecular mechanisms responsible. New technologies for rationally engineering proteases, as well as improved delivery options, will expand greatly the potential applications of these enzymes. The recognition that proteases are, in fact, an established class of safe and efficacious drugs will stimulate investigation of additional therapeutic applications for these enzymes. Proteases therefore have a bright future as a distinct therapeutic class with diverse clinical applications.     

防烟叶霉变菌株培养条件的研究

为有效防治烟叶霉变．对防烟叶霉变最好的3个菌株B112、B329、JMB142的发酵条件进行了初步探讨,通过测定吸光度值计算孢子浓度,确定了3个菌株生长的最适pH值与最适培养基。结果表明菌株JMB142在pH为7．2到8．4时,生长较好,菌株B112和329在PH6.3到PH7.9之间生长较好;当PH小于4.2时,三个菌株均不能正常生长,PH大于9.0时,三个菌株的生长都呈下降趋势。通过方差分析,比较了7种培养基对3株防烟叶霉变菌株产菌量的影响,结果表明,B112、JMB142菌株最佳培养基分别为6^#、5^#培养基,而5^#、7^#培养基更适合B329生长。