Effect of carbon and nitrogen sources on xylanase production by Trichoderma harzianum 1073 D3

In order to determine the effect of different carbon and nitrogen sources on xylanase production by Trichoderma harzianum 1073 D3, xylan in the xylanase production medium was replaced with different carbon sources. In order to reduce production time, glucose was added to the production media containing xylan. The effects of sucrose, maltose and lactose were investigated and maximum xylanase activity was observed in the presence of sucrose. Ammonium sulphate was the most appropriate inorganic nitrogen source for xylanase production and urea increased xylanase activity slightly.     

Self-fusion of protoplasts enhances chitinase production and biocontrol activity in Trichoderma harzianum

Protoplasts were isolated from Trichoderma harzianum strain PTh18 using lysing enzymes and self-fusion of T. harzianum protoplasts was carried out using polyethylene glycol in STC buffer. The fused protoplasts of T. harzianum were regenerated and 15 self-fusants were selected to study the chitinase production and biocontrol activity. High chitinase activity was measured in the culture filtrates of most of the self-fusants (87%) than the parent. Among the fusants, the strain SFTh8 produced maximum chitinase with a two-fold increase as compared to the parent strain. All the self-fusants exhibited increased antagonistic activity against Rhizoctonia solani than the parent. The crude chitinase preparation of SFTh8 lysed the mycelia of T. harzianum, Trichoderma viride and Trichoderma reesei and released the protoplasts in higher number than the crude chitinase preparation of parent strain PTh18.     

Regulation of chitinase synthesis in Trichoderma harzianum.

The production of chitinase by Trichoderma species is of interest in relation to their use in biocontrol and as a source of mycolytic enzymes. Fourteen isolates of the genus were screened to identify the most effective producer of chitinase. The best strain for chitinase was Trichoderma harzianum 39.1, and this was selected for study of the regulation of enzyme synthesis. Washed mycelium of T. harzianum 39.1 was incubated with a range of carbon sources. Chitinase synthesis was induced on chitin-containing medium, but repressed by glucose and N-acetylglucosamine. Production of the enzyme was optimal at a chitin concentration of 0.5%, at 28 degrees C, pH 6.0 and was independent of the age of the mycelium. The synthesis of chitinase was blocked by both 8-hydroxyquinoline and cycloheximide, inhibitors of RNA and protein synthesis, respectively. The mode of chitinase synthesis in this fungus is discussed.     

罗伊乳杆菌增殖培养基中碳源氮源的优化

目的提高罗伊乳杆菌的发酵活菌数,以提高发酵产率,降低生产成本。方法采用光电比浊法与活菌计数法,通过单因素试验和正交设计方法对罗伊乳杆菌的增殖培养基的碳源和氮源进行优化,并且绘制优化前后的生长曲线。结果罗伊乳杆菌增殖培养基中最佳的碳源、氮源种类与浓度为葡萄糖2.1%、蔗糖3.0%、牛肉膏1.5%、酵母粉1.4%,最佳的培养时间为10h。结论通过优化罗伊乳杆菌的增殖培养基,提高了发酵产率,降低了生产成本。     

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.     

Domestic wastewater as substrate for cellulase production by Trichoderma harzianum

Cellulase production using residues as substrate has been well described, as it is an interesting method of reducing the costs of processes, one of the main bottlenecks for the production of enzymes. This research describes for the first time the use of raw domestic wastewater, which is largely and continuously generated, as a culture base medium for cellulase production. The strain Trichoderma harzianum HBA03 was selected according to the highest activity produced for FPase (5.4 U/mL) and CMCase (8.2 U/mL). Peptone was selected as a nitrogen source and microcrystalline cellulose as the inducer for cellulase production, resulting in FPase activities of 5.6 and 5.0 U/mL and CMCase activities of 12.0 and 14.4 U/mL. The use of domestic wastewater as the culture medium led to an increase of 1.41 and 1.14 fold of FPase and CMCase production, respectively, compared to the synthetic medium. Production was also carried out in a bubble column reactor in which the maximum productivities achieved 10.2 U/L.h (FPase) and 64.6 U/L.h (CMCase). The presented results demonstrate the feasibility of the use of domestic wastewater for cellulases production, thereby contributing to the development of a sustainable process for reusing wastewater with a significant reduction in environmental impact.     

Rubratoxin production by penicillium rubrum when grown in a synthetic medium containing different sources of carbon and nitrogen

A sterile mineral salts broth was fortified with different additives, inoculated with conidia ofPenicillium rubrum P-13, and incubated quiescently for 14 days or with shaking for 3 to 5 days. Maximal fungal growth and rubratoxin production occurred when the broth contained 20% sucrose. Broth with 10% glucose, 10% fructose, 5% maltose, or 1% asparagine supported formation of substantial amounts of rubratoxin (52.9–78.5 mg/100 ml). When the broth was fortified with glucose plus lysine, arginine aspartic acid, cystine, ammonium citrate, or ammonium phosphate, moderate amounts (27.5–39.5 mg/100 ml) of rubratoxin and mycelium (0.1–1.5 g/100 ml) were produced. Presence in the broth of 5% galactose or starch resulted in accumulation of small amounts (22.2 and 24.6 mg/100 ml, respectively) of rubratoxin and mold tissue (0.70 and 0.5 g/ 100 ml, respectively). Whereas some toxin was recovered from mineral salts broth fortified with lactose or ribose, toxin was not recovered when the mold grew in broth containing mannitol or fumarate. With the exception of gluconate which supported some growth and toxin formation and ethanol which permitted formation of small amounts of toxin, other carbon sources resulted in little or no fungal growth and no toxin formation. Yields of rubratoxin decreased with an increase in amount of agitation or length of incubation ofP. rubrum cultures. Mold growth increased and toxin formation decreased with an increase in volume of culture.     

哈茨木霉几丁质酶cDNA基因的克隆及在酿酒酵母中的表达

为研究哈茨木霉 (Trichodermaharzianum)的生物防治机制并获得与生物防治相关基因 ,通过构建哈茨木霉菌丝生长期的cDNA文库及对部分表达序列标签序列的测定与生物信息学分析 ,成功获得了哈茨木霉几丁质酶v(ChiV)基因的全长cDNA序列。该基因的编码框长度为 1194bp ,编码 397个氨基酸 ,理论分子量为 4 4kD。将该基因构建到酿酒酵母诱导型表达载体pYES2上 ,转化到酿酒酵母H15 8菌株中 ,通过Northern杂交检验后 ,确定该基因在酿酒酵母转录水平上表达。在 β_半乳糖诱导下 ,转化子在培养 6 0h时产生的酶活活性最高 ,几丁质酶V最适活性温度为 37℃ ,在pH 6和pH 8时活性较高。     

Optimization of carbon and nitrogen sources for phytase production by Mitsuokella jalaludinii,a new rumen bacterial species

AIMS: The effects of different carbon and nitrogen sources on phytase production by Mitsuokella jalaludinii were evaluated and the optimization of rice bran (RB) and soybean milk (SM) concentrations in the medium for phytase production was also determined. METHODS AND RESULTS: Replacement of glucose, cellobiose and starch in MF1 medium by RB or palm kernel cake and replacement of trypticase peptone and yeast extract in the medium by SM or enzymatic digested soybean milk significantly increased the phytase production by M. jalaludinii. The optimal concentrations of RB and SM in the medium for phytase production were 15% RB and 20% SM or 20% RB and 10% SM or 20% RB and 20% SM and the phytase activities in the media were 12.53, 12.93 and 12.75 U g-1 culture broth, respectively. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: The high production of phytase by M. jalaludinii warrants further research to increase its yield by genetic manipulation for commercial application.     

Enhancement of cellulase production by Trichoderma viride using carbon/nitrogen double-fed-batch

Summary The production of cellulase by Trichoderma viride has been remarkably raised by means of a fed batch fermentation with the addition of cellulose as the carbon source and ammonia as nitrogen source during cultivation.     

Regulation of extracellular laccase production of Agaricus bisporus by nitrogen sources in the medium

Abstract The substrate specificity of cystathionine γ-synthase (EC 4.2.99.9) in various bacteria was examined. O-Succinyl- l -homoserine was used preferably as a substrate by facultative anaerobic Gram-negative bacteria belonging to such genera as Escherichia, Klebsiella, Enterobacter, Citrobacter, Erwinia, Serratia, Proteus and Salmonella . Among Gram-negative aerobic bacteria, bacteria belonging to the genera Pseudomonas, Xanthomonas and Alcaligenes also used O -succinyl- l -homoserine in preference to O -acetyl- l -homoserine. On the other hand, bacteria belonging to the genera Agrobacterium and Flavobacterium used O -acetyl- l -homoserine preferably. As to Gram-positive aerobic bacteria, bacteria of the genus Bacillus used O -acetyl- l -homoserine exclusively. Bacteria belonging to the genera Micrococcus, Corynebacterium, Brevibacterium and Arthrobacter used both O -acetyl- and O -succinyl- l -homoserine to similar extents.     

pH and thermal stability studies of chitinase from Trichoderma harzianum: A thermodynamic consideration

The combined effect of pH and temperature on chitinase was investigated using response surface methodology. A central composite design for two variables was employed. The optimal pH and temperature for the least degree of deactivation were found out to be 5.4 and 24°C respectively. The deactivation rate constants and the half life of chitinase were estimated at different pH and temperature combinations. At the optimal pH of 5.4, the rate of the deactivation was found to be the least. Thermodynamic parameters, viz., ΔH^*, ΔS^*, ΔG^* and activation energy of thermal deactivation of chitinase were calculated in the temperature range from 50°C to 60°C.     

Peroxidase production by carbon and nitrogen sources fed-batch culture of Arthromyces ramosus

Summary Carbon and nitrogen sources were investigated for improving peroxidase production by Arthromyces ramosus, a hyperproducer of peroxidase. Glucose as carbon source and a mixture of yeast extract and polypeptone at the ratio of 3 to 5 as nitrogen source in a production medium were shown to give the highest peroxidase activity. During the culture amino acids such as alanine, arginine, methionine, leucine, tyrosine and tryptophan were depleted. Therefore, glucose supplemented nitrogen source fed-batch culture was carried out and a peroxidase activity of 73 U/ml was obtained. This activity was 1.7 times higher than that of glucose fed-batch culture. This indicates that an adequate nitrogen source supply during the culture is effective for improving the peroxidase production by A. ramosus.     

Determination of some physiological factors affecting xylanase production from Trichoderma harzianum 1073 D3

In this study, different Trichoderma strains were tested and Trichoderma harzianum 1073 D3 was found to be the most potent xylanase producer. Then some cultural parameters, namely, incubation time, substrate concentration, initial culture pH and temperature were optimized in order to increase xylanase production from Trichoderma harzianum 1073 D3. The optimum incubation time was found to be 13 days. It was concluded that 1% xylan concentration is suitable for high xylanase production rate. The optimum temperature and pH were found to be 30 degrees C and 7, respectively. Also, it was determined that agitation during growth was suitable for efficient production.     

In vitro antagonism of cotton seedlings fungi and characterization of chitinase isozyme activities in Trichoderma harzianum

The antagonistic fungus Trichoderma harzianum is widely recognized as a potential biocontrol agent against several soil-borne plant pathogens. T. harzinum is rich source of chitinoltic enzymes. In vitro screening of 5 isolates of T. harzinum, one isolate of Chaetomium globosum and one isolate of Conetherium mentance, revealed that all of them had reduced growth area of Macrophomina phaseolina, Fusarium solaniand Rhizoctonia solani on PDA medium, significantly. The inhibition percentage ranged from 77.9 % to 55.9% for M. phaseolina and 59.2% to 40.4% for R. solani by T. harzinum and C. mentance, respectively. Inhibition for F. solani ranged from 76.5% to 55.7% by T. harzinum and C. globosum, respectively. Isozyme gel electrophoresis was used to assess chitinase activity secreted by selected isolates of T. harzinum under different pH degrees and temperatures. Obtained results indicated that activity of chitinase isozyme produced at 30 °C was higher than 15–20 °C for all tested isolates and activity of chitinase produced by isolates No. 4 and 5 of T. harzinum at pH (7–7.5) was higher than at pH 6, respectively.     

Optimization of carbon and nitrogen sources and growth factors for the production of an aquaculture probiotic (Pseudomonas MCCB 103) using response surface methodology

AIM: To develop a new medium for enhanced production of biomass of an aquaculture probiotic Pseudomonas MCCB 103 and its antagonistic phenazine compound, pyocyanin. METHODS AND RESULTS: Carbon and nitrogen sources and growth factors, such as amino acids and vitamins, were screened initially in a mineral medium for the biomass and antagonistic compound of Pseudomonas MCCB 103. The selected ingredients were further optimized using a full-factorial central composite design of the response surface methodology. The medium optimized as per the model for biomass contained mannitol (20 g l(-1)), glycerol (20 g l(-1)), sodium chloride (5 g l(-1)), urea (3.3 g l(-1)) and mineral salts solution (20 ml l(-1)), and the one optimized for the antagonistic compound contained mannitol (2 g l(-1)), glycerol (20 g l(-1)), sodium chloride (5.1 g l(-1)), urea (3.6 g l(-1)) and mineral salts solution (20 ml l(-1)). Subsequently, the model was validated experimentally with a biomass increase by 19% and fivefold increase of the antagonistic compound. CONCLUSION: Significant increase in the biomass and antagonistic compound production could be obtained in the new media. SIGNIFICANCE AND IMPACT OF THE STUDY: Media formulation and optimization are the primary steps involved in bioprocess technology, an attempt not made so far in the production of aquaculture probiotics.