Chitin degradation by Clostridium sp. strain 9.1 in mixed cultures with saccharolytic and sulfate-reducing bacteria

Abstract The fermentation of chitin was studied in pure and cocultures of the chitinolytic Clostridium strain 9.1 and various non-hydrolytic sugar-fermenting and sulfate-reducing bacteria. A 5- to 8-fold enhancement of the rate of chitin degradation was observed, which was not due to the alleviation of inhibition of the chitinolytic enzyme system by polymer hydrolysis products. This was concluded from the observation that rates of chitinolysis and fermentation were unaffected by the addition of N -acetylglucosamine (NAG) or NAG-oligomers to pure cultures of strain 9.1, and from the absence of an unequivocal relation between the ability of a secondary bacterium to consume potentially inhibitory hydrolysis products and its capacity to stimulate chitin degradation. The acceleration of chitin fermentation in the presence of sugar-fermenting bacteria was the result of a release by these secondary populations of growth factors essential to strain 9.1. These factors comprised a high molecular, thioredoxin-like compound responsible for enhanced chitinolytic activity [10], and various low molecular compounds necessary for optimal growth. The sulfate reducers (except Desulfovibrio sp. strain 20028) released primarily the high molecular growth factor in coculture with strain 9.1. NAG-fermenting bacteria consumed approximately 10% of the hydrolysis products, whereas species capable of utilizing both mono- and oligomeric sugars fermented at least 50% of the sugars produced by strain 9.1. Nevertheless, the rate of chitinolysis in these cocultures proceeded at very similar rates.
The observed interactions between Clostridium sp. strain 9.1 and the secondary populations are discussed in relation to the results from studies on mixed culture fermentations of cellulosic substrates reported in the literature.     

Peripheral enzymatic deacetylation of chitin and reprecipitated chitin particles

The enzymatic deacetylation of various chitin preparations was investigated using the fungal chitin deacetylase (CDA) isolated from Rhizopus oryzae growth medium. Specific extracellular enzyme activity after solid state fermentation was 10 times higher than that after submerged fermentation. Natural crystalline chitin is a very poor substrate for the enzyme, but showed a five-time better deacetylation after dissolution and reprecipitation. Chitin particles, enzymatically deacetylated for only 1% exhibited a strongly increased binding capacity towards ovalbumin, while maintaining the rigidity and insolubility of chitin in a moderate acidic environment. Because of the unique combination of properties, these CDA treated chitin materials were named "chit-in-osan". Chitinosan was shown to be an attractive matrix for column chromatography because no hydrogel formation was observed, that impaired the flow of eluent. Under the same conditions, partially deacetylated chitosan swelled and blocked the flow in the column.     

Kinetics of chitinase production. I. Chitin hydrolysis

As part of the development of a comprehensive mathematical model for chitinase production by Serratia marcescens QMB 1466 growing on chitin, the different mass transport and kinetic steps involved during chitin hydrolysis were studied. The experimental results for the hydrolysis of chitin by a crude preparation of chitinase show a system kinetically limited by the overall rate of chitin hydrolysis. This rate is linearly related to the concentration of enzyme adsorbed on the chitin particle. Adsorbed and bulk enzyme concentration were found to be related through a Langmuir type of isotherm.     

Mesophilic chitin-degrading anaerobes isolated from an estuarine environment

Abstract Eight strains of obligately anaerobic, mesophilic, chitinolytic bacteria were isolated from the sediment of an estuarine environment. The isolates were rod-shaped, Gram-negative, and formed terminal spherical spores that swelled the sporangium. The major products from the fermentation of chitin were: acetate, ethanol, formate, CO₂, H₂ and ammonia. Growth of the isolates was possible at pH values ranging from 5.0–9.0. During the fermentation of chitin, N -acetylglucosamine accumulated in the culture fluids and was not metabolized. No organic compounds other than chitin and its oligomers could be demonstrated to support growth of the isolates. Hydrolysis of chitin proceeded at a relatively low rate and was incomplete. Approximately 65% of the initial amount of chitin was hydrolyzed during a period of 5–10 days. Supplementation of the medium with yeast extract, casamino acids or peptone did not enhance the rate of chitin hydrolysis, but reducing agents such as Na₂S₂O₄ and Ti (III)-NTA markedly stimulated the rate of chitin fermentation.
The ecological implications of the high degree of substrate specialization are discussed.     

Estimation of fungal growth in a solid state fermentation system

Summary Of four chemical methods for estimating mycelial biomass in koji fermentation which were examined, the modified method of Ride and Drysdale, was found to be most suitable. The observed level of aldehyde, expressed as glucosamine, is related to fungal dry weight. The assay method correlates chitin levels with some enzyme activities in the fermentation. This method may be applicable for detecting the extent of fungal growth in other solid and semi-solid substrates.     

Biosynthesis of chitin by particulate fractions from Cunnighamella elegans.

The enzyme chitin synthetase (UDP-acetylaminodeoxyglucosyl transferase, EC 2.4.1.16) in Cunninghamella elegans has been investigated. The enzyme was present in the microsomal, cell wall, mitochondrial and the soluble cytoplasmic fraction of the mycelium, with the former having the highest specific activity. The properties of the enzyme in this fraction were investigated; the Km for UDP GlcNAc was 1.23 mM and 2.08 mM GlcNAc in the presence of 1 mM UDP GlcNAc. The temperature optimum was between 26 degrees and 29 degrees C and maximal activity was at pH 6.25. Mg++ ions had no effect on chitin synthesis, but soluble chitodextrins inhibited the enzyme. The production of chitin synthetase was correlated with the growth of the fungus, maximum activity being found during the late exponential phase of growth. Chitin was confirmed as the sole product of enzyme action, by digestion with chitinase.     

层生镰孢菌产甲壳素脱乙酰酶发酵动力学

对层生镰孢菌产甲壳素脱乙酰酶的发酵动力学进行了研究。通过Logistic方程分别构建层生镰孢菌细胞生长、甲壳素脱乙酰酶（CDA）合成及糖基质消耗的非结构动力学模型,并利用1stOpt软件对该模型进行了模拟,采用Origin8.0软件得到了非线性曲线拟合图形及各模型参数。结果表明,各模型预测值与实验数据能较好地拟合,层生镰孢菌细胞的比生长速率在第15.52h达到峰值（μ_{m, x}）0.160h^-1;层生镰孢菌的底物比消耗速率在26.51h时达到峰值（μ_{m, s}）0.096h^-1;层生镰孢菌的甲壳素脱乙酰酶比合成速率19.40h达到峰值（μ_{m, p}）0.548U/(mL·h)。模型拟合和实验数据具有良好的适应性,基本上反映了层生镰孢菌发酵产酶过程的动力学特征,为今后的工业化规模生产提供理论依据。     

Penicillium oxalicum SAEM-51: a mutagenised strain for enhanced production of chitin deacetylase for bioconversion to chitosan

A novel chitin deacetylase (CDA) producing strain Penicillium oxalicum ITCC 6965 was isolated from residual materials of sea food processing industries. Strain following mutagenesis using ethidium bromide (EtBr) and microwave irradiation had resulted into a mutant P. oxalicum SAE(M)-51 having improved levels of chitin deacetylase (210.71 ± 1.65 Ul(-1)) as compared to the wild type strain (108.26 ± 1.98 Ul(-1)). Maximum enzyme production was achieved in submerged fermentation following 144 hours of incubation with notably improved productivity of 1.46 ± 0.82 Ul(-1) h(-1) as compared to the wild type strain (0.75 ± 0.53 Ul(-1)h(-1)). Scanning electron micrographs of mutant and wild type strains had revealed distinct morphological features. Evaluation of kinetic parameters viz. Q(s), Q(p), Y(p/x), Y(p/s), q(p), q(s) had denoted that strain P. oxalicum SAE(M)-51 is a hyper producer of chitin deacetylase. Glucose as compared to chitin or colloidal chitin had resulted in increased levels of enzyme production. However, replacement of glucose with chitinous substrates had prolonged the duration for enzyme production. The mutant strain had two pH optima that is 6.0 and 8.0 and had an optimum temperature of 30 °C for growth and enzyme production.     

Study on the production of chitin and chitosan from shrimp shell by using Bacillus subtilis fermentation

Fermentation of shrimp shell in jaggery broth using Bacillus subtilis for the production of chitin and chitosan was investigated. It was found that B. subtilis produced sufficient quantities of acid to remove the minerals from the shell and to prevent spoilage organisms. The protease enzyme in Bacillus species was responsible for the deprotenisation of the shell. The pH, proteolytic activity, extent of demineralization and deprotenisation were studied during fermentation. About 84% of the protein and 72% of the minerals were removed from the shrimp shell after fermentation. Mild acid and alkali treatments were given to produce characteristic chitin and their concentrations were standardized. Chitin was converted to chitosan by N-deacetylation and the properties of chitin and chitosan were studied. FTIR spectral analysis of chitin and chitosan prepared by the process was carried out and compared with spectra of commercially available samples.     

裂褶菌F17固态发酵产锰过氧化物酶及其发酵动力学模型的建立

白腐真菌分泌的锰过氧化物酶是木质素降解酶系统的主要组分,对木质素解聚,纸浆和染料的脱色均有重要作用.利用裂褶菌F17在自行设计的通气托盘式反应器中,以松木屑、稻草及黄豆粉为混合营养基质进行固态发酵生产锰过氧化物酶.在自制通气托盘式反应器中,裂褶菌F17能够产生锰过氧化物酶,发酵96 h时,最高酶活力达到13.51 U/...     

Biosynthesis of endochitinase of Serratia marcescens.

The growth of a mutant strain of Serratia marcescens with high chitinase activity and the biosynthesis of endochitinase by this strain were investigated. The study was carried out using semisynthetic culture medium without inducers and culture medium containing colloidal chitin as a sole nitrogen and carbon source, with and without mitomycin C. The mutant strain, unlike the native one, was shown to produce endochitinase and to secrete the enzyme into the medium during the growth on culture medium without the inducers, chitin and mitomycin C. During growth on the medium with chitin the mutant strain differed from the native one with a short lag-phase of growth, the early appearance of endochitinase in the culture liquid and a high level of endochitinase activity. The difference between the strains disappeared after the addition of mitomycin C, an inducer of the cell SOS-response, to the culture medium containing chitin. Specific endochitinase activity of S. marcescens mutant strain grown on various culture media had two maxima, namely at the beginning and at the end of the stationary phase. Mitomycin C increased the specific activity in a second peak of endochitinase activity during the growth of the mutant strain.     

Interspecies interaction based on transfer of a thioredoxin-like compound in anaerobic chitin-degrading mixed cultures

Abstract Fermentation of chitin by mixed cultures of the chitinolytic Clostridium sp. strain 9.1 and various non-chitinolytic bacteria proceeded up to eight times faster than in pure cultures. The addition of spent media of such mixed cultures also resulted in a marked stimulation of chitinolysis in pure cultures of strain 9.1. Pure cultures fermented chitin much faster if supplemented with either spent media or cell-free extracts of the non-chitinolytic bacteria. The compound responsible for this stimulation was thermostable (10 min at 85° C) and could not be removed by passage over Sephadex G-25, indicating a molecular weight of more than 1500. The heat stable enzyme thioredoxin (from Saccharomyces cerevisiae ) was shown to stimulate the chitin fermentation in a similar manner. Alkylation of this enzyme reduced its stimulatory action significantly indicating its (di)thiol: disulfide interchanging activity.
It is hypothesized that essential sulfhydryl groups in the chitinolytic system of strain 9.1 are reduced by thioredoxin and/or similar thiol: disulfide transhydrogenases present in the cell-free extracts and spent media, resulting in an acceleration of chitin hydrolysis and fermentation. This stimulation may thus be the result of a new type of interspecies interaction in anaerobic mixed cultures.     

A pathway of chitosan formation in Mucor rouxii. Enzymatic deacetylation of chitin.

1. An enzyme that catalyzes hydrolysis of acetamido groups of chitin derivatives was found in the supernatant fraction of Mucor rouxii. 2. Partially O-hydroxyethylated chitin (glycol chitin) was used as a substrate in the purification and characterization of this enzyme. A 140-fold purification was obtained by means of ammonium sulfate fractionation followed by chromatography on carboxymethylcellulose and DEAE-cellulose. 3. The enzyme releases about 30% of the acetyl groups of glycol chitin, giving a product with a decreased sensitivity to lysozyme. The enzyme also deacetylates chitin and N-acetylchitooligoses, whereas it is inactive toward bacterial cell wall peptidoglycan, N-acetylated heparin, a polymer of N-acetylgalactosamine, di-N-acetylchitobiose and monomeric N-acetylglucosamine derivatives. 4. This enzyme shows a pH optimum of 5.5. The Km value for glycol chitin is 0.87 g/l or 2.6 mM with respect to monosaccharide residues. 5. The occurrence of this enzyme accounts for the formation of chitosan in fungi.     

Fermentation Kinetics and Continuous Process of L-Asparaginase Production

For the purpose of obtaining L-asparaginase in quantities from Erwinia aroideae, cell growth and enzyme formation were investigated in both batch and continuous fermentation. Using yeast extract as a growth-limiting substrate, the relationship between specific growth rate and substrate concentration was found to fit the Monod equation. The optimum temperature for enzyme production was 24 C, although cell growth was higher at 28 C. The enzyme yield reached its maximum of 4 IU/ml during the negative acceleration growth phase which occurs just prior to stationary growth. Compared to batch fermentations, the continuous fermentation process gave a lower enzyme yield except when the fermentation was conducted at a dilution rate of 0.1 hr(-1). The graphical method frequently used for prediction of continuous fermentation does not apply to L-asparaginase production by E. aroideae. The optimum temperature for enzyme production in continuous process was 24 C, which was the same as in batch process. Increasing the temperature from 24 to 28 C resulted in a 20% loss of enzyme yield.     

Influence of the carbon source on the growth and lignocellulolytic enzyme production by Morchella esculenta strains

Growth and lignocellulolytic enzymes production by two Morchella esculenta strains (BAFC 1728 and BEL 124) growing in solid state fermentation using different lignocellulosic materials along 58 days was characterized. Both strains were able to grow on the three substrates: wheat bran, wheat bran plus corn starch, and rolled oat. The growth was characterized by measuring chitin content, reducing sugars, pH, dry weight loss, and extractable proteins, such parameters varied substantially with substrate and strain used. The maximum rate of growth in both strains was observed between 5 and 28 days. Regarding enzyme production, as a general trend strain BAFC 1728 produced the highest titres. The most evident difference was observed in laccase production by this strain on wheat bran, which exceeded that observed in strain BEL 124 by tenfold (7.45 U g⁻¹).     

An enzyme kinetic model for describing fermentation processes

An enzyme kinetic model has been proposed to describe fermentation processes. This type of model was chosen because it is biologically sound, can incorporate all of the important engineering control variables, and can draw upon, in its development, the extensive kinetic literature. An intial qualitative test for this model was made on the gluconic acid fermentation. A necessary check of the model was that Monod's empirical cell growth and yield equations were derived as a special case. The model also offered an explanation for the hysteresis behavior of the gluconic acid production rate as a function of gluconolactone.     

Mathematical model for aerobic culture of a recombinant yeast

A mathematical model was formulated to simulate cell growth, plasmid loss and recombinant protein production during the aerobic culture of a recombinant yeast S. cerevisiae. Model development was based on three simplified metabolic events in the yeast: glucose fermentation, glucose oxidation and ethanol oxidation. Cell growth was expressed as a composite of these metabolic events. Their contributions to the total specific growth rate depended on the activities of the pacemaker enzyme pools of the individual pathways. The pacemaker enzyme pools were regulated by the specific glucose uptake rate. The effect of substrate concentrations on the specific growth rate was described by a modified Monod equation. It was assumed that recombinant protein formation is only associated with oxidative pathways. Plasmid loss kinetics was formulated based on segregational instability during cell division by assuming constant probability of plasmid loss. Experiments on batch fermentation of recombinant S. cerevisiae C468/pGAC9 (ATCC 20690), which expresses Aspergillus awamori glucoamylase gene and secretes glucoamylase into the extracellular medium, were carried out in an airlift bioreactor in order to evaluate the proposed model. The model successfully predicted the dynamics of cell growth, glucose consumption, ethanol metabolism, glucoamylase production and plasmid instability. Excellent agreement between model simulations and our experimental data was achieved. Using published experimental data, model agreement was also found for other recombinant yeast strains. In general, the proposed model appears to be useful for the design, scale-up, control and optimization of recombinant yeast bioprocesses.     

链霉菌A048产几丁质酶最佳发酵工艺研究

将链霉菌A048在完全培养基中培养至对数生长末期,离心洗涤收集菌丝体,然后接种入发酵产酶培养基中,进行二步发酵工艺牛产几丁质酶,几丁质酶活力比一步发酵工艺提高1．1倍,发酵周期共54h,比一步发酵工艺缩短66h;把菌丝体与几丁质粉共固定化,接入发酵产酶培养基中培养36h,几丁质酶活力比一步发酵工艺提高1．8倍,发酵周期缩短54h;在二步发酵工岂中另添加0．4％纤维素,几丁质酶活力可提高4倍,比一步发酵工艺提高10倍,酶活力达18．52U／mL。采用几丁质和纤维索双因子诱导二步发酵工艺可能是链霉菌A048生产几丁质酶的最佳工艺。     

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.     

Optimization of carbon and nitrogen sources in the medium and environmental factors for enhanced production of chitinase by Trichoderma harzianum

Statistical design was used to determine the optimal levels of medium components, the optimal initial pH of the enzyme production medium, the temperature of fermentation, age of the organism in the slant growth and the age of the inoculum for the production of chitinase in shake flask fermentations. The use of high concentrations of chitin and ammonium sulphate and exclusion of peptone and urea from the medium resulted in the production of higher level of the enzyme. The optimal concentrations of the medium components were 12.5 kg/m³ and 4.2 kg/m³ for the chitin and ammonium sulphate respectively. The effect of the addition of peptone and urea to the optimized medium was studied. The optimal values of initial pH and temperature were 5.6 and 28 °C respectively. The optimal age of the slant and the inoculum were found to be 105 h and 43 h respectively. The highest level of chitinase before optimization of the above variables was 0.054 U which was maximized to the level of 0.197 U.