Muramidase Catalyzed Hydrolysis of p-Nitrophenylacetate

It was found that muramidase can catalyze the hydrolysis of p-nitrophenylacetate (NPA) producing p-nitrophenol and acetic acid. The activity of muramidase for NPA, however, simply increased on raising a temperature and with an increase in alkalinity of reaction mixture. The mechanism of muramidase catalyzed hydrolysis of NPA differs from that of chymotrypsin which can catalyze burstly the hydrolysis of NPA by its histsdine residue.

The amount of reducing power produced owing to the hydrolysis of glycol chitin by muramidase was not affected by the presence of NPA, and inversely, the hydrolysis of NPA was not affected by glycol chitin. Obviously, there was no competitive inhibition between NPA and glycol chitin. The responses of modified muramidase to glycol chitin and to NPA did not correspond at all. The catalytic site of muramidase for glycol chitin may be different from that of muramidase for NPA. The hydrolysis of NPA is catalyzed by some free amino groups in the muramidase molecule, while the catalytic site for glycol chitin is not known.     

Purification and Some Properties of Cellulases from Aspergillus niger

Four types of cellulases, FI-1-b, FI-2-b, FI-2-c, and FII, were obtained from a commercial crude cellulase preparation produced from a water extract of a culture of A. niger.

Ammonium sulfate fractionation and column chromatography using DEAE-Sephadex A-25, Amberlite IRC-50 and Hydroxylapatite were employed for the purification of these cellulases.

Some properties of these enzymes were investigated: the optimum pH for the hydrolysis of glycol cellulose by FI-2-b and FI-2-c was pH 4.0 to 5.0, while that of FI-1-b was pH 2.3 to 2.5, and the optimum temperature for the activity of FI-2-b and FI-2-c was 40°C, but that of FI-1-b was 65°C.

The FII seemed to be most active toward cellobiose. Studies of the mode of action on glycol cellulose indicated that A. niger cellulases seemed not to be capable of attacking highly polymerized cellulose.     

SSF production,purification and characterization of chitin deacetylase from Aspergillus flavus

The production of an extracellular chitin deacetylase (CDA) produced by Aspergillus flavus under solid-substrate fermentation (SSF) using wheat bran as substrate was optimized using statistical methods. The CDA production in SSF increased 1.79-fold in comparison to the unoptimized basal level medium. It was purified to a final purity of 3.94-fold by ammonium sulphate precipitation, ion-exchange chromatography, and gel-permeation chromatography (GPC) consecutively and further characterized. The molecular mass of the enzyme was estimated to be about 28?kDa by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and GPC analysis. The optimum pH and temperature of the purified enzyme were pH 8.0 and 50?°C, respectively. Additionally, the effect of some cations and other chemical compounds on the CDA activity was studied. A marginal increase in enzyme activity was observed with metal ions mainly Mn²⁺ and Zn²⁺. No inhibition of the enzyme was observed by the end product, that is, acetate up to 70?mM concentration. The K_m and k_cat values of the enzyme were determined to be 9.45?mg mL^?1 and 26.72?s^?1 respectively, using colloidal chitin as substrate. Among various substrates tested, glycol chitin and colloidal chitin were deacetylated.     

Parameters optimization of chitin hydrolysis by Trichoderma harzianum chitinase under assay conditions

A kinetic analysis and optimization of reaction conditions for the enzymatic hydrolysis of chitin using chitinase produced by Trichoderma harzianum NCIM 1185 was carried out. Swollen chitin was used as the substrate for chitinase. The central composite design was followed for this optimization. The required volume ratio of the major reactants for maximum hydrolysis was determined. The pH and temperature optima were found to be 4.75 and 47 °C respectively. K _m and V _max for this enzyme were 4.643 kg/m³ and 0.1542 U respectively.     

Purification and Characterization of Extracellular Chitin Deacetylase from Colletotrichum lindemuthianum

Chitin deacetylase, active in the presence of acetate (96% of the enzymatic activity was retained in the presence of 100 mm sodium acetate), was purified to electrophoretic homogeneity from a culture filtrate of Colletotrichum lindemuthianum (944-fold with a recovery of 4.05%). The enzyme was induced in the medium after the eighth day of incubation simultaneously with the blackening of the medium. The molecular mass of the enzyme was 31.5 kDa and 33 kDa as judged by SDS–PAGE and gel filtration, respectively, suggesting that the enzyme is a single polypeptide. The optimum temperature was 60°C and the optimum pH was 11.5–12.0 when glycol chitin was used as substrate. The enzyme was active toward glycol chitin, partially N-deacetylated water soluble chitin, and chitin oligomers the degrees of polymerization of which were more than four, but was less active with chitin trimer and dimer, and inactive with N-acetylglucosamine. The K_m and k_cat for glycol chitin were 2.55 mm and 27.1s^?1, respectively, and those for chitin pentamer were 414 μm and 83.2s^?1, respectively. The reaction rates of the enzyme toward glycol chitin and chitin oligomers seemed to follow the Michaelis–Menten kinetics.     

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.     

Chitin and chitosan biopolymer production from the Iranian medicinal fungus Ganoderma lucidum: Optimization and characterization

Abstract

Chitin and chitosan with unique properties and numerous applications can be produced from fungus. The production of chitin and chitosan from the mycelia of an Iranian Ganoderma lucidum was studied to improve cell growth and chitin productivity. Inoculum size and initial pH as two effective variables on the growth of G. lucidum and chitin production were optimized using response surface method (RSM) by central composite design (CCD). The results verified the significant effect of these two variables on the cell growth and chitin production. In optimum conditions, including pH?=?5.7 and inoculum size of 7.4%, the cell dry weight was 5.91?g/L and the amount of chitin production was 1.08?g/L with the productivity of 0.083?g/(L day). The produced chitin and chitosan were characterized using XRD and FTIR. Moreover, the antibacterial activity of the produced chitosan was investigated and compared with the commercial chitosan. The results showed that the produced chitin and chitosan had suitable quality and the Iranian G. lucidum would be a great source for safe and high-quality chitin and chitosan production.     

Purification and characterization of a thermostable chitinase from <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> Mb-2

Summary A chitinase produced by Bacillus licheniformis MB-2 isolated from Tompaso geothermal springs, Indonesia, was purified and characterized. The extracellular enzyme was isolated by successive hydrophobic interaction, anion exchange, and gel filtration chromatographies. The purified enzyme was a monomer with an apparent molecular weight of 67 kDa. The optimal temperature and pH of the enzyme were 70 °C and 6.0, respectively. It was stable below 60 °C for 2 h and over a broad pH range of 4.0–11.0 for 4 h. The enzyme was resistant to denaturation by urea (1 M), Tween-20 (1%) and Triton-X (1%), but unstable toward organic solvents such as dimethyl sulphoxide, DMSO, (5%) and polyethylene glycol, PEG, (5%) for 30 min. The enzyme hydrolysed colloidal chitin, glycol chitin, chitosan, and glycol chitosan. The first 13 N-terminal amino acids of the enzyme were determined as SGKNYKIIGYYPS, which is identical to those in chitinases from B. licheniformis and B. circulans.     

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.     

Characterization of the First Fungal Glycosyl Hydrolase Family 19 Chitinase (NbchiA) from Nosema bombycis (Nb)

Chitinases (EC 3.2.1.14), as one kind of glycosyl hydrolase, hydrolyze the β‐(1,4) linkages of chitin. According to the sequence similarity, chitinases can be divided into glycoside hydrolase family 18 and family 19. Here, a chitinase from Nosema bombycis (NbchiA) was cloned and purified by metal affinity chromatography and molecular exclusion chromatography. Sequence analysis indicated that NbchiA belongs to glycoside hydrolase family 19 class IV chitinase. The optimal pH and temperature of NbchiA are 7.0 and 40 °C, respectively. This purified chitinase showed high activity toward soluble substrates such as ethylene glycol chitin and soluble chitosan. The degradation of chitin oligosaccharides (GlcNAc)_2–5 detected by high‐performance liquid chromatography showed that NbchiA hydrolyzed mainly the second glycosidic linkage from the reducing end of (GlcNAc)_3‐5. On the basis of structure‐based multiple‐sequence alignment, Glu51 and Glu60 are believed to be the key catalytic residues. The site‐directed mutation analysis revealed that the enzymatic activity was decreased upon mutation of Glu60, whereas mutation of Glu51 totally abolished the enzymatic activity. This is the first report of a GH19 chitinase in fungi and in Microsporidia.     

Endochitinase from Aspergillus nidulans implicated in the autolysis of its cell wall

An endochitinase from centrifuged autolyzed cultures of Aspergillus nidulans has been purified 100 times. The enzyme has Mw 27,000, pI of 4.8 units, pH optimum around 5 pH units. It is unstable at temperature greater than 70 degrees C and does not have a cation requirement. It is inhibited by Hg2+, Cu2+, Ca2+ and Ag+ and it does not have muramidase activity. The enzyme depolymerizes chitin rapidly with production of high molecular weight polysaccharides, and then slowly degrades these with production of N,N'-diacetylchitobiose. The enzyme hydrolyzes N,N',N'-triacetylchitotriose with production of N,N'-diacetylchitobiose and N-acetylglucosamine and this hydrolysis is inhibited by other chitin oligomers and N-acetylglucosamine. This enzyme hydrolyzes in the same way the chitin obtained from the cell wall of Aspergillus nidulans.     

Repeated Enzymatic Hydrolysis of Polygalacturonic Acid,Chitosan and Chitin Using a Novel Reversibly-soluble Pectinase with the Aid of κ-carrageenan

Aspergillus niger pectinase, together with κ-carrageenan, could be precipitated in the presence of 0.2% KCl and re-dissolved by ten-fold dilution of the salt. The free as well as this reversibly-soluble (rs) enzyme were evaluated for hydrolysis of polygalacturonic acid, chitosan and chitin. The rs-enzyme showed 92%, 80% and 74% activity (as compared to the corresponding amount of enzyme when present as a free enzyme) towards the three substrates, respectively. There was no significant change in the pH and temperature optima of the rs-enzyme. This preparation could be reused six times without loss of any detectable polygalacturonase activity. This biocatalyst design was found to be efficient for the hydrolysis of polygalacturonic acid, chitosan and chitin.     

Purification and characterization of a Bacillus circulans No. 4.1 chitinase expressed in Escherichia coli

Summary A DNA fragment encoding for 598 amino acids of chitinase protein from Bacillus circulans No. 4.1 was subcloned into pQE-30 expression vector and transformed into Escherichia coli M15 (pREP4). The molecular weight of the expressed protein was approximately 66 kDa. Enzymatic activity of the recombinant protein was assayed after purification using affinity chromatography on a nickel chelating resin. The enzyme hydrolyzed N-acetylchitooligosaccharides mainly to N-acetylchitobiose, and was active toward chitin, carboxymethyl-chitin, colloidal chitin, glycol chitin and 4-methylumbelliferyl-β-d-N, N′-diacetylchitobiose. The pH and temperature optima of the chitinase enzyme were 7.0 and 45 °C, respectively. This enzyme was stable in the pH range of 5.0–9.0 and at temperatures up to 50 °C. In addition, when cleaved by a proteolytic enzyme, the 20-kDa product could retain high chitinolytic activity.     

An extracellular Bacillus sp. chitinase for the production of chitotriose as a major chitinolytic product

An extracellular chitinase of Bacillus sp. WY22 was purified by 9.6-fold. It had a Mr of 35 kDa, an apparent K _m value for colloidal chitin of 3 mg ml^–1 and was optimally active at 37 °C and pH 5.5 over 1 h. The enzyme could also hydrolyse swollen chitin, glycol chitin and chitosan with relative activities of 76%, 34% and 23% compared with colloidal chitin. It formed chitotriose as a major product from colloidal chitin and glycol chitin.     

Characterization of Chitinase Produced by the Alkaliphilic <Emphasis Type="Italic">Bacillus mannanilyticus</Emphasis> IB-OR17 B1 Strain

The paper reports on the isolation of an extracellular chitinase produced by the alkaliphilic Bacillus mannanilyticus IB-OR17 B1 strain grown in media containing crab shell and bee chitin at a pH of 8–11. The enzyme was 860-fold purified by ultrafiltration and chitin sorption. The molecular weight of the purified chitinase was shown by denaturing electrophoresis to be 56 kDa. The enzyme showed maximum activity at a pH of 7.5–8.0 and 65°C and was stable within a pH range of 3.5–10.5 and temperature range of 75–85°C. With colloidal chitin as substrate, the kinetic characteristics of the chitinase were determined as follows: K_M ~ 1.32 mg/mL and V_max ~ 5.05 μM min^–1. N-acetyl-D-glucosamine and its dimer were the main products of enzymatic chitin cleavage, while the trisaccharide was detected just in minor quantities. The chitinase actively hydrolyzed p-nitrophenyl-GlcNAc₂ according to the exo-mechanism of substrate hydrolysis characteristic of chitobiosidases.     

Chemical and Enzymatic Properties of Acid-cellulase Produced by Aspergillus niger

Some properties of a purified acid-cellulase produced by Aspergillus niger were investigated. The acid-cellulase was stable at the pH range between 4.0 and 10.0 and exhibited the highest activity toward glycol cellulose at pH 2.5. The optimum temperature of activity was measured to be 50 C, while the enzyme was inactivated above 40‘C by heating for 1 hr. Insoluble cellulose such as filter paper was difficult to be attacked by the enzyme.

Mg²⁺ and Mn²⁺ ions inhibited the activity, while Co²⁺ ion caused a slight activation.

The nitrogen content of the enzyme protein was determined to be 14.37%. The enzyme contained 378 residues of amino acids rich in acidic amino acids, 12 residues of glucosamine and 10 residues of arabinose per molecule. N-terminus was not detected by DNP-method.     

Conversion of the enzymatic hydrolysate of shellfish waste chitin to single-cell protein

The conversion of the enzymatic hydrolysate of shellfish chitin waste to single-cell protein was investigated as part of a comprehensive waste treatment program. Forty-two yeasts were screened for ability to assimilate the monomer of chitin, N-acetylglucosamine, which has been shown to be the sole product of enzymatic hydrolysis of chitin. The Yeast Pichia Kudriavzevii was selected for study, based on ability to grow at high temperature (37°C and above), low pH (4.0 ± 0.5), and in a nutritionally simple medium. Growth rates of P. kudriavzevii were similar on N-acetylglucosamine and on the chitin hydrolysate. Dependencies of specific growth rate on temperature, pH, medium composition, and oxygen tension were studied. The variations of yield, protein content, and total nucleic acid content with the specific growth rate were evaluated. The amino acid distribution of the protein of P. kudriavzevii was obtained.     

Direct fermentation of potato starch wastewater to lactic acid by Rhizopus oryzae and Rhizopus arrhizus

The biochemical kinetic of direct fermentation for lactic acid production by fungal species of Rhizopus arrhizus 3,6017 and Rhizopus oryzae 2,062 was studied with respect to growth pH, temperature and substrate. The direct fermentation was characterized by starch hydrolysis, accumulation of reducing sugar, and production of lactic acid and fungal biomass. Starch hydrolysis, reducing sugar accumulation, biomass formation and lactic acid production were affected with the variations in pH, temperature, and starch source and concentration. A growth condition with starch concentration approximately 20 g/l at pH 6.0 and 30°C was favourable for both starch saccharification and lactic acid fermentation, resulting in lactic acid yield of 0.87–0.97 g/g starch associated with 1.5–2.0 g/l fungal biomass produced in 36 h fermentation. R. arrhizus 3,6017 had a higher capacity to produce lactic acid, while R. oryzae 2,062 produced more fungal biomass under similar conditions.     

Proteinase inhibitor synthesis in tomato leaves : induction by chitosan oligomers and chemically modified chitosan and chitin

Soluble chemical derivatives of chitin and chitosan including ethylene glycol chitin, nitrous acid-modified chitosan, glycol chitosan, and chitosan oligomers, produced from chitosan by limited hydrolysis with HCl, were found to possess proteinase inhibitor inducing activities when supplied to young excised tomato (Lycopersicon esculentum var Bonnie Best) plants. Nitrous acid-modified chitosans and ethylene glycol chitin exhibited about 2 to 3 times the activity of acid hydrolyzed chitosan and 15 times more activity than glycol chitosan. The parent chitin and chitosans are insoluble in water or neutral buffers and cannot be assayed. Glucosamine and its oligomers from degree of polymerization = 2 through degree of polymerization = 6 were purified from acid-fragmented chitosan and assayed. The monomer was inactive and dimer and trimer exhibited weak activities. Tetramer possessed higher activity and the larger pentamer and hexamer oligomers were nearly as active as the total hydrolyzed mixture. None of the fragments exhibited more than 2% acetylation (the limits of detection). The contents of the acid-fragmented mixture of oligomers was chemically N-acetylated to levels of 13% and 20% and assayed. The N-acetylation neither inhibited nor enhanced the proteinase inhibitor inducing activity of the mixture. These results, along with recent findings by others that chitinases and chitosanases are present in plants, provide further evidence for a possible role of soluble chitosan fragments as signals to activate plant defense responses.     

Pentane Inhibition of the Egg-white Lysozyme-induced Cell Lysis of Micrococcus lysodeikticus

Pentane inhibited the cell lysis of Micrococcus lysodeikticus by egg-white lysozyme, when added to the lysozyme solution before mixing the cells. The pentane inhibition was not observed when pentane was added either before to the cell suspensions or to the cell-lysozyme mixture. The degree of pentane inhibition was proportional to the concentration of pentane, and the maximum inhibition was achieved with about 5% pentane close to the saturation point. On the other hand, pentane did not inhibit the hydrolysis of glycol chitin by the lysozyme, showing that the β-1,4-glucosaminidase activity of lysozyme remained unchanged. The inhibitory action of pentane on the lysozyme-induced cell lysis was of a competitive nature. The pentane inhibition had no ph-dependence, but it was influenced by the ionic strength of the buffer used as solvent.

When the lysozyme solution was treated with pentane, a characteristic ultraviolet spectrum of lysozyme was produced; a blue shift with a minimum at 280 nm and a trough at 291 nm. The degree of spectral change at 280 nm depended on the concentration of pentane. Pentane was, therefore, found to interact with egg-white lysozyme to affect the conformation and enzymatic activity of lysozyme. The mechanism of pentane inhibition on the lysozyme-induced cell lysis was discussed.