nitro-N-nitrosoguanidine Research Articles

Mutagenesis development of actinoplanes sp. KCTC 9161 by N-methyl-N'-nitro-N-nitrosoguanidine (NTG) and screening for acarbose production

Acarbose has been widely used in the therapy of type II diabetes (non-insulin dependent) because it controls blood sugar contents of patients after meals. Acarbose, a pseudo-oligosaccharide, acts as a competitive -glucosidase inhibitor. Acarbose is produced by the strains of Bacillus, Streptomyces and Actinoplanes sp. The aim of this study was to develop mutagenesis for an Actinoplanes sp. strain and screening for acarbose production. The spores of Actinoplanes sp. KCTC 9161 strain were subjected to be mutated by N-methyl-N'-nitro-N-nitrosoguanidine (NTG) for screening and finding mutant strains that were capable of production of higher acarbose (an inhibitor of α-glucosidase) higher than wild type strain. Firstly, the original NTG solution was prepared in phosphate buffer 0.05 M, pH 6.9 and the safety concentration of NTG was determined at 5 mg/ml. Then, the spores were incubated with different NTG amounts and duration. The living colonies were transferred to fermentation medium. The results obtained showed that 15 mutant strains were produced higher acarbose than wild type when used thin layer chromatography method for analysis and comparing with standard acarbose (Sigma). Three cell lines among total tested 15 mutant lines of Actinoplanes sp. KCTC 9161 produced acarbose at a higher level or indicated a higher inhibitory activity toward α-glucosidase than the original strain. Enzymatic inhibitory ativity of α-glucosidase of three mutant strains (Actinoplanes sp. KCTC- L4, L11, L14) was increased 1.3 fold higher than wild type and Actinoplanes sp. KCTC spores were very sensitive to NTG toxic, 98% spores could not survive at the treatment condition of 50 µg NTG for 30 minutes. In addition, an applicable protocol for mutating Actinoplanes sp. using NTG was suggested for further research.

Highly efficient production of hyaluronic acid by Streptococcus zooepidemicus R42 derived from heterologous expression of bacterial haemoglobin and mutant selection.

During Streptococcus zooepidemicus fermentation, most carbon sources are used to synthesize lactic acid, which can inhibit strain growth and hyaluronic acid production. Here, we expressed bacterial haemoglobin (Vhb) in Strep.zooepidemicus. Due to highly efficient oxygen use, only 15·26gl(-1) lactic acid was produced, which is 0·73 times the quantity produced by the control strain. Compared with the control strain (1·61gl(-1) ), hyaluronic acid (HA) production in this strain did not substantially increase, only to 2·16gl(-1) . Next, we used a series of N-methyl-N'-nitro-N-nitroso-guanidine (NTG) treatments and selection programmes. Finally, we generated a hyaluronidase-negative and rifampin-resistant mutant strain that produces high levels of HA. The optimum carbon concentration for maximum hyaluronic acid production is only 30gl(-1) of sucrose, which is lower than the control strain (60gl(-1) ). The oxygen transfer rate coefficient KL a increased significantly to 372±53h(-1) from 18±4h(-1) of the control. The optimum carbon source for this strain is 21gl(-1) of sucrose, 9gl(-1) of maltose and 5gl(-1) of glutamic acid. Hyaluronic acid accumulated at 6·7gl(-1) in the culture broth. However, the molecular weight of HA decreased from 1835KDa (Control) to 429kDa. The prepared low-molecular weight HA could function as potential antiangiogenic substances, antiviral and antitumour agents to possibly be used as functional food ingredients. Hyaluronic acid (HA) has been used for a wide range of applications in health, cosmetic and clinical fields. During fermentation of Streptococcus to produce HA, 80-85% of the carbon source is used to produce lactic acid and acetic acid, and only approx. 5 and 10% of the carbon source is used to produce HA and biomass respectively. Here, we expressed bacteria haemoglobin (Vhb) in Streptococcus zooepidemicus, which can dramatically inhibit lactic acid production. After NTG treatments and selection programmes, we identified a mutant strain with highly efficient hyaluronic acid production (6·7gl(-1) ) under economic fermentation conditions.

English

Many traditional mutagenic strategies have been used to improve cellulase production by micro-organisms, especially fungi species. Trichoderma species are among cellulolytic fungi, those that have been most extensively studied, due to their efficient production of these enzymes. In the present study, N-methyl-N´-nitro-N-nitrosoguanidine (NTG) was used as mutagenic agent to obtain cellulolytic mutant from wild strain T. atroviride 676. After mutagenic procedures, two strains (102C1 and 104C2) were selected as promising cellulase-producing mutant. The effect of the carbon (sugarcane bagasse: SCB) and nitrogen (corn steep liquor: CSL) sources on endoglucanase production by the mutants 102C1 and 104C2 was studied using submerged cultivations at 28°C. Different concentrations of SCB and CSL were used and nine different media were generated. Mutant 102C1 showed the best results when using 2.5% SCB and 0.7% CSL. A central composite rotational design (CCRD) was performed to estimate optimal conditions of pH and temperature for endoglucanase activity of strain 102C1, which were pH 3.6 and temperature 66°C. The characterization of this acidophilic and thermophilic endoglucanase activity produced by the mutant strain 102C1 allows its use in biotechnological applications, particularly in the hydrolysis of agro industrial residues, such as SCB, for bioethanol production.   Key words: Trichoderma atroviride, CMCase, nitrosoguanidine, sugarcane bagasse, corn steep liquor.

Biosynthesis of isocitric acid by the yeast yarrowia lipolytica and its regulation

We studied the biosynthesis of isocitric acid from rapeseed (canola) oil by the yeast Yarrowia lipolytica and its regulation. We determined a fundamental possibility for directed biosynthesis of isocitric acid by Y lipolytica yeast, with only minimal amounts of citric acid byproduct, when grown on a medium containing canola oil. Wild type strains of Y lipolytica were mutagenized by UV irradiation and treatment with N-methyl-N'-nitro-N-nitrosoguanidine (NG). Subsequent selection on media with acetate and isocitrate resulted in isolation of a UV/NG Y lipolytica UV/NG mutant that synthesized isocitrate and citrate at a ratio of 2.7:1. In the parent strain, this ratio is 1:1. Inhibition of isocitrate lyase, a key enzyme in the metabolism of isocitric acid, by the addition of itaconic acid resulted in increased synthesis of isocitrate with a ratio of isocitrate to citrate reaching 6:1. Culturing of the Y lipolytica UV/NG mutant in a pilot industrial fermenter in the presence of itaconic acid resulted in the production of 88.7 g/L of isocitric acid with a yield of 90%.

Optimization and strain improvement by mutation for enhanced cellulase production by Bacillus sp. (MTCC10046) isolated from cow dung

The goal was to investigate the cellulase enzyme production ability of bacterial strain C1 isolated from cow dung and identified as Bacillus sp. on the basis of 16 S rDNA sequence homology. The effects of different carbon sources like Carboxymethyl cellulose (CMC), avicel, starch, maltose, sucrose, glucose, fructose, galactose and lactose on cellulase production at varying environmental parameters of incubation period (2–10days), temperature (35–55°C), and pH (6.0–8.5) were examined. The CMC was the best carbon source for cellulase production followed by lactose in this bacterial strain. The maximum enzyme production was achieved at a temperature of 50°C by Bacillus sp. with pH of 7.0 on the 8th day of growth. The nitrogen source NH4NO3 at 0.175% was optimum for cellulase production by this bacterium. A putative mutant (C1M26) was screened from wild C1 strain after mutagenesis with N-methyl-N′-nitro-N-nitrosoguanidine (NTG) as a mutagenic agent. The mutant C1M26 produced a larger amount of cellulase in comparison to wild type C1 strain.

Enhanced Lycopene Content in Blakeslea trispora by Effective Mutation-Screening Method

The zygomycete fungus Blakeslea trispora is usually used as a natural source of lycopene and β-carotene. In this study, the B. trispora (-) strain, a major mating type for lycopene production, was treated with N(+) ion implantation and N-methyl-N'-nitro-N-nitrosoguanidine (NTG), and further isolated on the screening plates supplemented with lovastatin and crude extracts of trisporic acid (CTA). After several rounds of screening, four mutants with higher yield of lycopene and biomass were isolated. Among these mutants, I5 obtained with N(+) ion implantation showed a maximum lycopene yield (28.8 mg/g), which was 64 % higher than the parent strain (17.5 mg/g) in the production of lycopene. The results indicated that N(+) ion implantation is more suitable for B. trispora (-) than NTG treatment, and the addition of lovastatin promoted the generation of positive mutant and CTA amplified the color differences between colonies.

IMPROVEMENT OF MICROBIAL STRAIN AND FERMENTATION PROCESS OF RAPAMYCIN BIOSYNTHESIS

The purpose of this investigation is to enhance the production of the immunosuppressant drug rapamycin by subjecting the strain CBS 773.23 to ultraviolet (UV) and N-methyl-N′-nitro-N-nitroso guanidine (NTG) mutations. Among all the mutants tested, MTCC 5681 (NRC-CM03/SH) obtained by NTG mutagenesis of strain CBS 773.72 showed the highest activity, 210 mg/L. The effect of different factors including medium composition, pH, temperature, and intensity of mixing on rapamycin production was studied. Based on the study, the optimal concentrations of soluble starch and dry yeast granules were found to be 50 g/L and 1.5 g/L, respectively. Furthermore, optimal values for pH, temperature, and shaking speed were found to be 6.0, 28°C, and 220 rpm, respectively. The production of rapamycin increased 1.6-fold, to 360 mg/L, in shake-flask culture using the optimal combination of factors observed compared with basal cultivation medium using MTCC 5681 mutant strain.

The production of polyhydroxyalkanoate by Bacillus licheniformis using sequential mutagenesis and optimization

The purpose of this study was to enhance the production of polyhydroxyalkanoate (PHA) by sequential mutation of Bacillus licheniformis PHAs-007, using UV and N-methyl-N′-nitro-N-nitrosoguanidine (NTG). In addition, the effect of nutrient additions and environmental conditions were optimized to increase the production of PHA. Bacillus licheniformis PHAs-007 produced high amounts of PHA (64.09 ∼ 68.80% of DCW) under both synthetic and renewable substrates. After mutagenesis treatment, mutant M2-12 was selected from 380 strains, based on its high biomass and PHA concentration. The mutant M2-12 gave the highest value of specific growth rate (0.09/h), biomass (22.24 g/L) and PHA content (19.55 g/L) under optimal conditions, consisting of 3% palm oil mill effluent, with no additional trace elements, at 45oC and pH 7. The mutant strain showed higher resistance to substrate concentrations, as well as pH and temperature, than the wild type. The accumulation of PHA was increased by 3.18-fold compared to the wild type, and the production of PHA by the mutant M2-12 was constantly retained over 12 times of cultivation. The mutation and optimization strategy appear to be suitable for producing high density PHA, reducing the medium cost and consequently lowering the production cost. Interestingly, the mutant strain could synthesize the novel PHA copolymers such as 3-hydroxyvalerate and 3-hydroxyhexanoate, which were not produced by the wild type.

Over-expression of gluconic acid in Aspergillus oryzae RP-21 mutants generated by a random mutagenesis approach

Random mutagenesis with N-methyl-N′ nitro-N-nitrosoguanidine (NTG) was used to mutate Aspergillus oryzae RP-21 to develop high gluconic acid-producing mutants. Forty mutant colonies (designated as A. oryzae strains RP-NTG-01 to RP-NTG-40) screened for gluconic acid, glucose dehydrogenase and glucose oxidase production using a 12-well plate method showed that 17 strains (positive mutants) produced high concentrations of these three products, whereas 12 strains (negative mutants) showed low concentrations and the remaining 11 strains (non-mutants) did not produce any of the three products. Detailed studies of A. oryzae RP-NTG-12, a positive mutant, produced gluconic acid of up to 72 g/L in batch fermentation, which was a 2.4-fold increase in yield to that of the strain and as expected it also possessed higher activities of cell-bound glucose dehydrogenase and glucose oxidase, key enzymes of the multi-functional gluconic acid synthesis pathway. We discuss changes in the cell-bound enzyme activities of the mutants and the wild type and speculate on a mechanism for this increase. The mutant strain, A. oryzae RP-NTG-12, and the random mutagenesis method used to increase bioproducts have a good potential for developing fermentation processes to an industrial scale as demonstrated by this study.

Enhanced cellulose production by ultraviolet (UV) irradiation and N-methyl-N'-nitro-N-nitrosoguanidine (NTG) mutagenesis of an Acetobacter species isolate

Cellulose-producing bacteria were isolated from 14 bacterial cellulose (BC) factories in Thailand, and subjected to ultraviolet (UV) irradiation and/or N-methyl-N'-nitro-N-nitrosoguanidine (NTG) mutagenesis for strain improvement. BC yields of the NTG mutant NU4-NTG30-51 were 54.68% higher than that of the reference strain Acetobacter xylinum ATCC 10245 and 43.69% higher than that of the parent strain NU4 (P < 0.01). BC yields of the UV mutant NU4-UV40-07 were 50.59% higher than that of A. xylinum and 39.60% higher than that of NU4. Glucose consumption of these mutants was also higher than that of NU4. However, crystallinity of cellulose produced by NU4-NTG30-51 was higher than that produced by NU4-UV40-07. Combining UV irradiation and NTG did not result in BC yields higher than those of the mutants generated with a single mutagen. The mutant strains were then tested for reversion. Our results demonstrate that the BC yields of these mutants remained higher than that of the reference strain A. xylinum ATCC10245, and did not revert to the same level as the parent strain. The isolate NU4 and the two mutants were identified as Acetobacter species.

Systematic mutagenesis method for enhanced production of bacitracin by Bacillus licheniformis mutant strain UV-MN-HN-6

The purpose of the current study was intended to obtain the enhanced production of bacitracin by Bacillus licheniformis through random mutagenesis and optimization of various parameters. Several isolates of Bacillus licheniformis were isolated from local habitat and isolate designated as GP-35 produced maximum bacitracin production (14±0.72 IU ml-1). Bacitracin production of Bacillus licheniformis GP-35 was increased to 23±0.69 IU ml-1 after treatment with ultraviolet (UV) radiations. Similarly, treatment of vegetative cells of GP-35 with chemicals like N-methyl N’-nitro N-nitroso guanidine (MNNG) and Nitrous acid (HNO2) increased the bacitracin production to a level of 31±1.35 IU ml-1 and 27±0.89 IU ml-1respectively. Treatment of isolate GP-35 with combined effect of UV and chemical treatment yield significantly higher titers of bacitracin with maximum bacitracin production of 41.6±0.92 IU ml-1. Production of bacitracin was further enhanced (59.1±1.35 IU ml-1) by optimization of different parameters like phosphate sources, organic acids as well as temperature and pH. An increase of 4.22 fold in the production of bacitracin after mutagenesis and optimization of various parameters was achieved in comparison to wild type. Mutant strain was highly stable and produced consistent yield of bacitracin even after 15 generations. On the basis of kinetic variables, notably Yp/s (IU/g substrate), Yp/x (IU/g cells), Yx/s(g/g), Yp/s, mutant strain B. licheniformis UV-MN-HN-6 was found to be a hyperproducer of bacitracin.

Genome-wide analysis of mutagenesis bias and context sensitivity of N-methyl-N′-nitro-N-nitrosoguanidine (NTG)

We have analyzed the mutation spectrum of N-methyl-N′-nitro-N-nitrosoguanidine (NTG) from a set of 4099 mutations identified from whole-genome sequencing of 32 E. coli strains mutagenized with NTG. These data permit precise measurement of NTG's bias for G/C to A/T transitions (96.6% of all mutations) and also show that NTG mutagenesis is strongly sensitive to context, favoring guanine residues preceded by purines by five-fold over those preceded by pyrimidines. These data give confident estimates for the GC bias and transition/transversion ratios of NTG mutagenesis, which could not be estimated confidently from previous, much smaller datasets.

Generation of a catR deficient mutant of P. putida KT2440 that produces cis, cis-muconate from benzoate at high rate and yield

Pseudomonas putida KT2440-JD1 was derived from P. putida KT2440 after N-methyl-N'-nitro-N-nitrosoguanidine (NTG)-mutagenesis and exposure to 3-fluorobenzoate (3-FB). The mutant was no longer able to grow using benzoate as a sole carbon source, but co-metabolized benzoate to cis, cis-muconate during growth on glucose, which accumulated in the growth medium. The specific production rate (q(pm)) was 0.18±0.03 g cis, cis-muconate/(g(DCW) h) in continuous cultures, and increased to 1.4 g cis, cis-muconate/(g(DCW) h) during wash-out cultivation. Transcriptome analysis showed that the cat operon was not induced in P. putida KT2440-JD1 in the presence of 5mM benzoate, due to a point mutation in the highly conserved DNA binding domain of the transcriptional regulator (catR) of the cat operon. The ben operon was highly expressed in the presence of benzoate in the mutant and its parental strain. This operon contains PP_3166 (catA2), which was shown to be a second catechol 1,2-dioxygenase besides catA. P. putida KT2440-JD1 is the first cis, cis-muconate-accumulating mutant that was characterized at the genetic level. The specific production rate achieved is at least eight times higher than those reported for other cis, cis-muconate-producing strains.

Enhanced Incorporation of 3-Hydroxy-4-Methylvalerate Unit into Biosynthetic Polyhydroxyalkanoate Using Leucine as a Precursor

Ralstonia eutropha PHB-4 expressing Pseudomonas sp. 61-3 polyhydroxyalkanoate (PHA) synthase 1 (PhaC1Ps) synthesizes PHA copolymer containing 3-hydroxybutyrate (3HB) and a small amount (0.5 mol%) of 3-hydroxy-4-methylvalerate (3H4MV) from fructose as a carbon source. In this study, enhanced incorporation of 3H4MV into PHA was investigated using branched amino acid leucine as a precursor of 3H4MV. Leucine has the same carbon backbone as 3H4MV and is expected to be a natural and self-producible precursor. We found that the incorporation of 3H4MV was enhanced by the supplementation of excess amount (10 g/L) of leucine in the culture medium. This finding indicates that 3H4MV can be derived from leucine. To increase metabolic flux to leucine biosynthesis in the host strain by eliminating the feedback inhibition, the cells were subjected to N-methyl-N'-nitro-N-nitrosoguanidine (NTG) mutagenesis and leucine analog resistant mutants were generated. The mutants showed statistically higher 3H4MV fraction than the parent strain without supplementing leucine. Additionally, by supplying excess amount of leucine, the mutants synthesized 3HB-based PHA copolymer containing 3.1 mol% 3H4MV and 1.2 mol% 3-hydroxyvalerate (3HV) as minor constituents, which significantly affected the thermal properties of the copolymer. This study demonstrates that it is possible to enhance the monomer supply of 3H4MV into PHA by manipulating leucine metabolism.

Enhanced l-phenylalanine production by recombinant Escherichia coli BR-42 (pAP-B03) resistant to bacteriophage BP-1 via a two-stage feeding approach

The L-phenylalanine (L-Phe) production by Escherichia coli WSH-Z06 (pAP-B03) was frequently prevented by bacteriophage BP-1 infestation. To cope with the bacteriophage BP-1 problem for an improved L-Phe production, one bacteriophage BP-1-resistant mutant, E. coli BR-42, was obtained from 416 mutant colonies of E. coli WSH-Z06 after N-methyl-N'-nitro-N-nitrosoguanidine (NTG) mutagenesis by selection for resistance to bacteriophage BP-1. The recombinant E. coli BR-42-carrying plasmid pAP-B03 had a high capacity in L-Phe production and a remarkable tolerance to 1 × 10(10) pfu (plaque-forming unit)/ml bacteriophage stock. For an enhanced L-Phe production by E. coli BR-42 (pAP-B03), the effects of different feeding strategies including pH-stat, constant rate feeding, linear decreasing rate feeding, and exponential feeding on L-Phe production were investigated; and a two-stage feeding strategy, namely exponential feeding at μ (set) = 0.18 h(-1) in the first 20 h and a following linear varying rate feeding with F = (-0.55 × t + 18.6) ml/h, was developed to improve L-Phe production. With this two-stage feeding approach, a maximum L-Phe titer of 57.63 g/l with a high L-Phe productivity (1.15 g/l/h) was achieved, which was 15% higher than the highest level (50 g/l) reported so far according to our knowledge. The recombinant E. coli BR-42 (pAP-B03) is a potential L-Phe over-producer in substantial prevention of bacteriophage BP-1 infestation compared to its parent strain WSH-Z06 (pAP-B03).

High-level production of erythritol by mutants of osmophilic Moniliella sp.

Abstract An erythritol-producing osmophilic yeast-like fungus, Moniliella sp. 440, was isolated from honey and then successively mutated with iterative rounds of N-methyl-N′-nitro-N-nitrosoguanidine (NTG) treatment and selection. Six generations of mutants, named N12115-6, N21105-6, N31074-3, N42208-2, N53199-9, and N61188-12, were selected for and produced erythritol at 151.0, 157.2, 177.8, 191.4, 196.6, and 237.8 g/L, respectively, while the wild type strain produced 113.0 g/L erythritol in media containing 40% glucose and 1% yeast extract. The mutant cells were found to have a short rod-like shape, while the wild type cells have a long rod-like shape. The most efficient erythritol producer, N61188-12, assimilated myo-inositol and weakly assimilated erythritol. However, the wild type strain did not assimilate myo-inositol and assimilated erythritol well. In 250-L and 2000-L pilot-scale fermentors, the erythritol production by N61188-12 was 151.4 g/L and 152.4 g/L, respectively. A simple fed-batch culture of strain N61188-12 in a 2000-L fermentor increased erythritol production to 189.4 g/L after 10 days fermentation.

Enhanced l-phenylalanine biosynthesis by co-expression of pheA fbr and aroF wt

A β-2-thienylalanine-resistant E. coli K12 mutant carrying a Thr326Pro mutation in the regulation (R) domain of pheA ( pheA fbr ) was obtained by N-methyl-N′-nitro-N-nitrosoguanidine (NTG) mutagenesis. In the presence of 200 mM l-phenylalanine ( l-Phe), a recombinant E. coli WSH-Z06 (pAP-B03) carrying pheA fbr as well as wild-type aroF ( aroF wt ) exhibited more than 70% of the chorismate mutase-prephenate dehydratase (CM–PDT) activity as observed in the absence of this amino acid. The l-Phe titer of WSH-Z06 (pAP-B03) reached 35.38 g/L in a 3-L fermentor, which was 2.81-fold higher than that of the original strain E. coli WSH-Z06. Furthermore, the l-Phe yield on glucose of WSH-Z06 (pAP-B03) (0.26 mol/mol) was twice that of E. coli WSH-Z06. This recombinant E. coli WSH-Z06 (pAP-B03) is a potential strain for over-production of l-Phe.

Indigenous and Environmental Modulation of Frequencies of Mutation in Lactobacillus plantarum

Reliability of microbial (starter) strains in terms of quality, functional properties, growth performance, and robustness is essential for industrial applications. In an industrial fermentation process, the bacterium should be able to successfully withstand various adverse conditions during processing, such as acid, osmotic, temperature, and oxidative stresses. Besides the evolved defense mechanisms, stress-induced mutations participate in adaptive evolution for survival under stress conditions. However, this may lead to accumulation of mutant strains, which may be accompanied by loss of desired functional properties. Defining the effects of specific fermentation or processing conditions on the mutation frequency is an important step toward preventing loss of genome integrity and maintaining the productivity of industrial strains. Therefore, a set of Lactobacillus plantarum mutator reporter strains suitable for qualitative and quantitative analysis of low-frequency mutation events was developed. The mutation reporter system constructed was validated by using chemical mutagenesis (N-methyl-N'-nitro-N-nitrosoguanidine) and by controlled expression of endogenous candidate mutator genes (e.g., a truncated derivative of the L. plantarum hexA gene). Growth at different temperatures, under low-pH conditions, at high salt concentrations, or under starvation conditions did not have a significant effect on the mutation frequency. However, incubation with sublethal levels of hydrogen peroxide resulted in a 100-fold increase in the mutation frequency compared to the background mutation frequency. Importantly, when cells of L. plantarum were adapted to 42 degrees C prior to treatment with sublethal levels of hydrogen peroxide, there was a 10-fold increase in survival after peroxide treatment, and there was a concomitant 50-fold decrease in the mutation frequency. These results show that specific environmental conditions encountered by bacteria may significantly influence the genetic stability of strains, while protection against mutagenic conditions may be obtained by pretreatment of cultures with other, nonmutagenic stress conditions.

Strain improvement of Trichoderma reesei Rut C-30 for increased cellulase production.

The strain of Trichoderma reesei Rut C-30 was subjected to mutation after treatment with N-methyl-N'-nitro-N-nitrosoguanidine (NG) for 6 h followed by UV irradiation for 15 min. Successive mutants showed enhanced cellulase production, clear hydrolysis zone and rapid growth on Avicel-containing plate. Particularly, the mutant NU-6 showed approximately two-fold increases in activity of both FPA and CMCase in shake flask culture when grown on basal medium containing peptone (1%) and wheat bran (1%). The enzyme production was further optimized using eight different media. When a mixture of lactose and yeast cream was used as cellulase inducer, the mutant NU-6 yielded the highest enzyme and cell production with a FPase activity of 6.2 U ml(-1), a CMCase activity of 54.2 U ml(-1), a β-glucosidase activity of 0.39 U ml(-1), and a fungal biomass of 12.6 mg ml(-1). It deserved noting that the mutant NU-6 also secreted large amounts of xylanases (291.3 U ml(-1)). These results suggested that NU-6 should be an attractive producer for both cellulose and xylanase production.

Screening of asporogenic mutants of phytase-producingAspergillus nigerCFR 335 strain

Objective: Aspergillus niger is one of the fungi exploited industrially for the production of various metabolites including the enzyme phytase. Intensive sporulation is a serious crisis in using this fungus, hence, in the present study an attempt was made to obtain an asporulating strain. Materials and methods : A. niger CFR 335, a local phytase-producing isolate, was induced to physical mutation by ultraviolet (UV) radiation and chemical mutation using different concentrations of ethyl methane sulphonate (EMS) and N-methyl N?-nitro N-nitrosoguanidine (NTG) .Results: UV exposure for 30 min, 10 mM EMS and 25 ug ml^-1 of NTG were found to be optimum for maximum killing effect of 26.3%, 95% and 80% of the spores, respectively. In this experiment, a stable auxotrophic mutant (ala) of A. niger CFR 335 with suppressed sporulation was obtained. The auxotrophic mutant M-1 (A. niger CFR 335 ala) showed an improved specific activity of 1.26 U mg^-1 as compared with the parent strain (0.85 U mg1). Conclusion : Sporulation was drastically reduced in the mutant M-1 without affecting phytase yield, whereas the production rate was proportional to the rate of sporulation in the parent strain (A. niger CFR 335). Key words: asporulation, Aspergillus niger CFR 335, phytase, ethyl methane sulphonate, N- methyl N?-nitro N-nitrosoguanidine, ultraviolet radiation