Methionine Yield Research Articles

The Effect of Inoculum Size and Medium Volume on Methionine Production by Bacillus species EZ-13 and ZM-10

The effect of inoculum size and medium volume on methionine production by Bacillus species EZ- 13 and ZM-10 was studied. The methionine-producing bacteria had already been isolated from within Anambra State University (now Chukwuemeka Odumegwu Ojukwu University), Uli campus Anambra State. They were purified and identified as Bacillus species EZ-13 and ZM-10 using cultural and biochemical characteristics. Thereafter, they were used to evaluated the influence of inoculum size and medium volume on methionine production by Bacillus species EZ-13 and ZM-10 in 250 ml flasks containing 50 ml of sterile fermentation medium. The findings revealed that Bacillus species EZ-13 and ZM-10, at inoculum size of 4% v/v, recorded maximum methionine yields of 2.8 mg/ml and 2.1 mg/ml respectively. The methionine concentrations of both Bacillus species decreased beyond inoculum size of 4% v/v. The medium volume of 50 ml stimulated maximum methionine yield of 3.0 mg/ml in Bacillus species EZ-13 and 2.4 mg/ml in Bacillus species ZM-10. The methionine concentrations for both bacteria decreased beyond medium volume of 50 ml. The result of the study showed that varying the inoculum sizes and medium volumes, could influence methionine yield by fermentation.

Methionine Production and Optimization Using Bacillus cereus Isolated From Soil

Fermentation processes have become a common practice for overproduction of amino acids nowadays as it is cheaper and easier than other processes for commercial production of methionine and other essential amino acids. Methionine is a sulfur-containing essential amino acid required in the diet of humans and other animals for normal growth and functions of the body. It is widely used in feed, pharmaceutical and food industries. The research was focused on fermentative production of Methionine by Bacillus cereus isolated from soil. Methionine was produced under optimized conditions (Temperature, pH, Incubation time and agitation rate) using one factor at a time (OFAT). Optimum methionine yield of (4.520 mg/mL) was obtained at temperature of 35oC, pH of 7.5, and agitation rate of 125 rpm and incubation time of 96 h using OFAT. Characterization of methionine produced using Bacillus cereus shows similar properties with the commercially produced methionine by using Fourier Transform Infrared Spectroscopy (FTIR) which shows the amino and carbonyl group spectrum at 1640 cm-1 and 3257 cm-1 respectively. Thin Layer Chromatography (TLC) showed a band of methionine produced by bacillus cereus aligned with the commercial methionine.

Utilization of Various Starch Hydrolysates and Defatted Proteins by Bacillus cereus for Microbial Synthesis of Methionine in Submerged Medium

Three of the methionine-producing bacteria previously isolated from different soil ecovars in Owerri, characterized as Bacillus cereus no: DS13, RS16 and AS9, based on 16s rRNA sequencing, were screened for utilization agricultural products for production of methionine. Experimental studies on the Bacillus strains were carried out to examine the use of various starch hydrolysates from different agricultural products: wheat, sorghum, cassava, cocoyam, yam, plantain, millet, corn, potato, rice and defatted plant proteins: pigeon pea, cowpea, bambaranut, groundnut and soybean meal as source of carbon and nitrogen, respectively for the production of methionine. The result of the influence of various carbon and nitrogen sources on methionine production showed that 2.32mg/ml methionine accumulation in the culture broth of B. cereus DS13 when corn-starch hydrolysate/defatted groundnut meal was used, while B. cereus RS16 and Bacillus cereus DS13 gave the same methionine yield of 2.22mg/ml with corn-starch hydrolysate/defatted groundnut meal and sorghum hydrolysate/soyabean meal respectively. Bacillus cereus AS9 utilized wheat starch hydrolysate/soaybean meal, followed by plaintain-starch hydrolysate/bambaranut to produced 1.79mg/ml and 1.78mg/ml methionine respectively. Methionine was accumulated in the fermentation broth of the Bacillus strains after a fermentation period of 96h. This study indicates the possible utilization of agricultural products as substrates for L-methionine production in submerged medium especially when considering the limitations of producing methionine by fermentation processes.

INTERACTION OF DZR1, OPAQUE-2 AND NORMAL ENDOSPERM MAIZE INBRED LINES FOR GRAIN YIELD AND PROTEIN QUALITY

The objectives of this study were to (i) investigate genetic relationships among high lysine (HK) (opaque-2), high methionine (HM) (dzr1) and high yielding maize inbred lines (ii) to evaluate grain yield and protein quality of hybrids produced from these germplasm groups. Fifty-six hybrids generated from an 8 × 8 full diallel mating design were tested at two locations in Turkey in 2017 and 2018. Significant reciprocal effects for lysine, methionine, lysine quality index and methionine quality index revealed that parent effects may not be ignored in breeding for these traits. Lower grain yield among crosses produced from non-normal endosperm suggested that for high yield and improved amino acid concentration at least one parent of HK or HM should be used in hybrid combinations. HM × HM hybrids were not only good for high methionine, but also high lysine and protein. Several of the experimental hybrids in this study outperformed the commercial checks in terms of lysine and methionine yield. M2 × S1 hybrid had 27 % more lysine (58.6 kg ha-1) and 26 % more methionine (42.5 kg ha-1) yield than commercial high yielding normal hybrids. This study revealed that HK and HM germplasm can be combined with adapted high yield maize inbred lines to develop high yielding, high methionine and high lysine hybrids.

Lysine Production of <i>Microbacterium lacticum</i> by Submerged Fermentation Using Various Hydrocarbon, Sugar and Nitrogen Sources

Bacterial isolation from oil-contaminated and uncontaminated soil was screened for hydrocarbon utilizer which was also capable of producing lysine. Microbial production of lysine by Microbacterium lacticum was investigated in submerged fermentations using various concentrations of hydrocarbon, sugar sources and nitrogen. Of the nine sugar and five nitrogen sources tested, glucose/ammonium sulphate proved optimum for lysine production. Effect of varying concentration of carbon and nitrogen sources on lysine accumulation showed that glucose (4%) ammonium sulphate (1%) respectively increased lysine production. A gram positive rod bacterium identified as Microbacterium lacticum was identified. Optimizing the cultural conditions of Microbacterium lacticum in submerged medium gave a methionine yield of 2.99 mg/ml lysine in the broth culture after 96 h.

Improved Cultural Conditions for Methionine Accumulation in Submerged Cultivation of Bacillus cereus S8

Aims: To improve the cultural conditions for enhanced methionine production by Bacillus cereus S8 Study design: Study of the fermentation process in shake flask culture. Place and Duration of Study: Department of Applied Microbiology and Brewing, Nnamdi Azikwe University, Awka, Nigeria between 2011 to 2012. Methodology: The effects of medium/fermenter volume ratio, carbon and nitrogen sources, growth stimulators, vitamins and amino acid on methionine accumulation in the broth culture of Bacillus cereus S8 were investigated. The time course for methionine production was also studied. Results: A 20% medium/fermenter volume ratio improved methionine yield. Glucose and ammonium sulphate at 6.0 and 1.0% respectively stimulated methionine accumulation by Bacillus cereus S8. Yeast extract, peptone, DL-leucine and all vitamins studied enhanced methionine production. A methionine yield of 3.23mg/ml was produced after 96h fermentation and at a pH of 6.90. Conclusion: Improving the cultural conditions of Bacillus cereus S8 in submerged medium stimulated methionine increase. Original Research Article British Microbiology Research Journal, 4(8): 885-895, 2014 886

Optimization of Fermentation Conditions of <i>Bacillus thuringiensis</i> EC1 for Enhanced Methionine Production

Bacillus thuringiensis EC1, isolated from the fermented oil bean seed, Pentachletra macrophila Benthan, produced a methionine yield of 1.89 mg/ml. The influence of cultural conditions on methionine accumulation by B. thuringiensis EC1 showed that a 20% medium/fermenter volume ratio and a 5% inoculum size increased methionine yield. The carbon of choice was maltose and at 8% level stimulated methionine production. Among the nitrogen sources studied, ammonium sulphate was found to be the best and at 1% concentration produced a methionine yield of 2.56 mg/ml. All growth-promoting substances and their mixtures enhanced methionine accumulation by B. thuringiensis EC1. The effect of Vitamins on methionine production showed that riboflavin and thiamine HCl at 1.0 μg/ml yielded 2.49 mg/ml and 2.80 mg/ml methionine respectively. The influence of bivalent metals on methionine accumulation indicated that Zn2+ at all concentration stimulated methionine production. Mg2+ and Ba2+ at 0.1 μg/ml and 10.0 μg/ml respectively improved methionine yield. Optimizing the cultural conditions of B. thuringiensis EC1 in submerged medium gave a methionine yield of 3.18 mg/ml.

Influence of Antibiotics and Surfactants Addition on Growth and Methionine Productivity by <i>Bacillus cereus</i>

The effects of antibiotics and surfactant addition on methionine accumulation in the broth culture of three strains of methionine yeilding Bacillus cereus (B. cereus DS-13, B. cereus AS-9 and B. cereus RS-16) recovered from different soil ecovars in Owerri Nigeria were investigated. Ampicillin and erythromycin neither stimulated growth nor enhanced methionine production in Bacillus cereus DS-13, while tetracycline and chloramphenicol stimulated growth and enhanced methionine yield in Bacillus cereus DS-13 and Bacillus cereus AS-9. Ampicllin had the highest stimulatory effect on growth and methionine production in Bacillus cereus RS 16. Tween 80 and palmitic acid stimulated growth and improved methionine accumulation in the broth culture of all Bacillus strains while stearic acid did not improve yield in all Bacillus strains.

Towards methionine overproduction in Corynebacterium glutamicum - methanethiol and dimethyldisulfide as reduced sulfur sources

In the present work, methanethiol and dimethyldisulfide were investigated as sulfur source for methionine synthesis in Corynebacterium glutamicum. In silico pathway analysis has predicted a high methionine yield for these reduced compounds provided that they can be utilized. Wild type cells were able to grow on methanethiol and on dimethyldisulfide as sole sulfur source, respectively. Isotope labeling studies with mutant strains exhibiting targeted modification of methionine biosynthesis gave detailed insight into the underlying pathways involved in assimilation of methanethiol and dimethyldisulfide. Both sulfur compounds are incorporated as entire molecule, adding the terminal S-CH3 group to O-acetylhomoserine. In this reaction, methionine is directly formed. MetY (O-acetylhomoserine sulfhydrylase) was identified as enzyme catalyzing this reaction. Deletion of metY resulted in methionine auxotrophic strains grown on methanethiol or dimethyldisulfide as sole sulfur source. Plasmid based overexpression of metY in the delta metY background restored the capability to grow on methanethiol or dimethyldisulfide as sole sulfur source. In vitro studies with the C. glutamicum wild type revealed a relatively low activity of MetY for methanethiol (63 mU/mg) and dimethyldisulfide (61 mU/mg). Overexpression of metY increased the in vitro activity to 1780 mU/mg and was beneficial for methionine production, since the intracellular methionine pool was increased two-fold in the engineered strain. This positive effect was limited by depletion of the metY substrate O-acetylhomoserine, requesting for further metabolic engineering targets towards competitive production strains.

The response of slow-growing chicks to the supplementations with different methionine levels and/or two types of enzymes

In order to study productive performance and meat quality of slow-growing chicks used for rural poultry production in relation to methionine and enzyme supplementations, basal diets were formulated to contain 0.32, 0.29 and 0.27% methionine levels from 1 to 42 d of age, 43 to 84 d of age, and 85 to 98 d of age, respectively. These diets were supplemented with 0, 0.06 and 0.12% of DL-methionine; thus, there were three methionine levels during each period. These levels were fed with or without 750 FTY of phytase-6 or one g/kg of multienzymes; thus there were three methionine levels by three enzyme treatments in factorial design. Methionine levels of 0.44, 0.41 and 0.33% significantly increased productive performance from 1 to 42, 43 to 84 and 85 to 98 d of age, respectively. Phytase supplemented-basal diet and diet supplemented with 0.06% methionine and phytase yield similar productive performance to those of chicks fed diet supplemented with 0.06% and 0.12% methionine, respectively. Phytase improved significantly nutrient digestibilities, percentage dressed carcass and chest parts compared to the control and multienzymes groups. Increasing methionine levels by 0.06 and 0.12% improved significantly percentage dressed carcass, chest and hind parts by 2.2 and 2.7, 1.4% and 2.8 and 3.1 and 2.5%, respectively. However, the methionine level and/or type of enzyme supplementations did not significantly affect metabolic profiles such as plasma total protein, albumen, globulin, total lipids and cholesterol.

Metabolic pathway analysis for rational design of L-methionine production by Escherichia coli and Corynebacterium glutamicum

Metabolic pathway analysis was carried out to predict the metabolic potential of Corynebacterium glutamicum and Escherichia coli for the production of L-methionine. Based on detailed stoichiometric models for these organisms, this allowed the calculation of the theoretically optimal methionine yield and related metabolic fluxes for various scenarios involving different mutants and process conditions. The theoretical optimal methionine yield on the substrates glucose, sulfate and ammonia for the wildtype of C. glutamicum is 0.49 (C-mol) (C-mol) −1, whereas the E. coli wildtype exhibits an even higher potential of 0.52 (C-mol) (C-mol) −1. Both strains showed completely different optimal flux distributions. C. glutamicum has a high flux through the pentose phosphate pathway (PPP), whereas the TCA cycle flux is very low. Additionally, it recruits a metabolic cycle, which involves 2-oxoglutarate and glutamate. In contrast, E. coli does minimize the flux through the PPP, and the flux through the TCA cycle is high. The improved potential of the E. coli wildtype is due to its membrane-bound transhydrogenase and its glycine cleavage system as shown by additional simulations with theoretical mutants. A key point for maximizing methionine yield is the choice of the sulfur source. Replacing sulfate by thiosulfate or sulfide increased the maximal theoretical yield in C. glutamicum up to 0.68 (C-mol) (C-mol) −1. A further increase is possible by the application of additional C 1 sources. The highest theoretical potential was obtained for C. glutamicum applying methanethiol as combined source for C 1 carbon and sulfur (0.91 (C-mol) (C-mol) −1). Substrate requirement for maintenance purposes reduces theoretical methionine yields. In the case of sulfide used as sulfur source a maintenance requirement of 9.2 mmol ATP g −1 h −1, as was observed under stress conditions, would reduce the maximum theoretical yield from 67.8% to 47% at a methionine production rate of 0.65 mmol g −1 h −1. The enormous capability of both organisms encourages the development of biotechnological methionine production, whereby the use of metabolic pathway analysis, as shown, provides valuable advice for future strategies in strain and process improvement.

Production of methionine by a multi-analogue resistant mutant of Corynebacterium lilium

Multi-analogue resistant mutants of Corynebacterium lilium were developed using UV and N-methyl- N′-nitro-nitrosoguanidine (NTG) mutagenesis in order to overproduce methionine. Although the methionine yield at each stage of mutation by UV and NTG mutagenesis were comparable, it was observed that the NTG mutants had higher cell growth and glucose utilization rates. From the parent NTG mutant C. lilium E 4 resistant to ethionine, the highest methionine producing strain C. lilium M-128 resistant to 3.5 mg ml −1 of ethionine, 3.0 mg ml −1 of norleucine, 3.8 mg ml −1 of methionine sulphoxide and 3.8 mg ml −1 of methionine methyl sulphoniumchloride was derived. When C. lilium M-128 was grown under optimised conditions, it produced 2.3 g l −1 of methionine in a 15 l batch reactor. The maximum cell mass concentration obtained was 17 g l −1 dry cell weight resulting in a cell yield coefficient of 0.34 g g −1. The dissolved oxygen concentration was controlled at 40% saturation by cascade control of the airflow rate and agitation speed.

Biotechnological production of high specific activity L‐35 S‐cysteine and L‐35 S‐methionine by using a diploid yeast Saccharomyces cerevisiae

AbstractHigh specific activity L‐35 S‐cysteine and L‐35 S‐methionine were synthesised by using a wild type diploid strain of baker's yeast—Saccharomyces cerevisiae. Yeast cells were grown in a sulphur depleted synthetic medium in which Na2 35 SO4 (50 mCi/ml) was supplemented as the sole sulphur source. The level of incorporation was 60% on an average. The protein hydrolysate of the cultured cells was subjected to paper and column chromatographic separations to get the individual L‐35 S‐aminoacids. The radiochemical yields of cysteine and methionine were 6–7% and 18–20% respectively. The radiochemical purity of the products was >95%. The highest specific activity for the products obtained by employing this method was 1100 Ci/mmole from the starting material, Na2 35 SO4, with a specific activity of 1350 Ci/mmole.

Enhancement of methionine production by methionine analogue ethionine resistant mutants of Brevibacterium heali

AbstractIn order to improve the methionine yield of the isolate B. heali, attempts were made to isolate mutants resistant to the methionine analogue DL‐ethionine after mutagenesis with N‐methyl‐N′‐nitro‐N‐nitrosoguanidine (NTG). The minimum inhibitory concentration (MIC) of ethionine for B. heali was found to be 2 mM. After mutagenesis and screening, five mutants resistant to 50 mM of ethionine were isolated. The yield of the best ethionine resistant mutant, B. heali Br EthR, was 13 mg/l of methionine medium under optimum cultivation conditions.

Recovery of tryptophan from 25-minute acid hydrolysates of protein

It was found that thioglycolic acid prevents destruction of tryptophan during rapid hydrolysis of protein with a trifluoroacetic acid HCl mixture (1:2, v v ) at 166°C for 25 or 50 min. The addition of 5% ( v v ) thioglycolic acid gave the maximum tryptophan recovery (88.3%) for a 25-min hydrolysate of lysozyme. Tryptophan recoveries varied slightly among three different proteins; 88% for lysozyme, 73% for α-chymotrypsinogen A, and 85% for apomyoglobin. However, when extrapolated to zero time, the values were close to one another: 94, 87, and 88%, respectively. The addition of thioglycolic acid was also advantageous for recovering amino acids other than tryptophan. Particularly, yields of carboxymethylcysteine and methionine were greatly improved. This modified rapid hydrolysis method gave satisfactory results without the need for separate analyses of tryptophan and cysteine, provided proteins were reduced and carboxymethylated prior to hydrolysis.

Oxidation of tryptophan to oxindolylalanine by dimethyl sulfoxide-hydrochloric acid. Selective modification of tryptophan containing peptides.

Tryptophan is readily oxidized to oxindolylalanine (2-hydroxytryptophan) in good yield on treatment in acetic acid solution with a mixture of dimethyl sulfoxide (DMSO) and concentrated aqueous HCl at room temperature. Other sulfoxides can be used in combination with HCl; for example, methionine sulfoxide reacts with an equimolar amount of tryptophan to give high yields of methionine and oxindolylalanine. Methionine and cysteine are quantitatively oxidized by DMSO/HCl to methionine sulfoxide and cystine, respectively. The tryptophan containing peptides LRF (luteinizing hormone-releasing factor), somatostatin, valine-gramicidin A and ACTH 1-24 were each treated with the DMSO/HCl reagent in acetic acid solution and the corresponding oxindolylalanine-derivatives isolated in over 90% yield after chromatography. The identity and purity of the derivatives were established on the basis of ultraviolet spectral characteristics and quantitative amino acid analysis of the oxindolylalanine content of acid hydrolyzates of the oxidized peptides with 3N-p-toluenesulfonic acid at 110 degrees for 24 h. The results indicate that modification of tryptophan peptides with DMSO/HCl provides a useful procedure, which seems superior to previously used reagents. In addition, the method could be well applied to other indoles of biological and pharmacological interest.