Showing papers in "Biotechnology Letters in 2007"
TL;DR: These studies will help in designing a rational enzymatic strategy for the synthesis of nanomaterials of different chemical composition, shapes and sizes as well as their separation.
Abstract: Synthesis of silver nanoparticles using α-NADPH-dependent nitrate reductase and phytochelatin in vitro has been demonstrated for the first time. The silver ions were reduced in the presence of nitrate reductase, leading to the formation of a stable silver hydrosol 10–25 nm diam. and stabilized by the capping peptide. The nanoparticles were characterized by X-ray diffraction, transmission electron microscopy, X-ray photoelectron spectroscopy and UV-Vis absorption. These studies will help in designing a rational enzymatic strategy for the synthesis of nanomaterials of different chemical composition, shapes and sizes as well as their separation.
566 citations
TL;DR: This review focuses on the past, current and future development of various experimental protocols in the rapid developing area of metabolomics in the ongoing quest to reliably quantify microbial metabolites formed under defined physiological conditions.
Abstract: Microbial metabolomics has received much attention in recent years mainly because it supports and complements a wide range of microbial research areas from new drug discovery efforts to metabolic engineering. Broadly, the term metabolomics refers to the comprehensive (qualitative and quantitative) analysis of the complete set of all low molecular weight metabolites present in and around growing cells at a given time during their growth or production cycle. This review focuses on the past, current and future development of various experimental protocols in the rapid developing area of metabolomics in the ongoing quest to reliably quantify microbial metabolites formed under defined physiological conditions. These developments range from rapid sample collection, instant quenching of microbial metabolic activity, extraction of the relevant intracellular metabolites as well as quantification of these metabolites using enzyme based and or modern high tech hyphenated analytical protocols, mainly chromatographic techniques coupled to mass spectrometry (LC-MSn, GC-MSn, CE-MSn), where n indicates the number of tandem mass spectrometry, and nuclear magnetic resonance spectroscopy (NMR).
353 citations
TL;DR: This article summarizes efforts in industry and academia to use TGE to produce tens to hundreds of milligrams of r-proteins for either fundamental research or preclinical studies.
Abstract: The expansion of the biologics pipeline depends on the identification of candidate proteins for clinical trials Speed is one of the critical issues, and the rapid production of high quality, research-grade material for preclinical studies by transient gene expression (TGE) is addressing this factor in an impressive way: following DNA transfection, the production phase for TGE is usually 2-10 days Recombinant proteins (r-proteins) produced by TGE can therefore enter the drug development and screening process in a very short time--weeks With "classical" approaches to protein expression from mammalian cells, it takes months to establish a productive host cell line This article summarizes efforts in industry and academia to use TGE to produce tens to hundreds of milligrams of r-proteins for either fundamental research or preclinical studies
298 citations
TL;DR: CO2 at different concentrations were added to cultures of the eukaryotic microalgae, Chlorella kessleri, C. vulgaris and Scenedesmus obliquus, and the prokaryotic cyanobacterium, Spirulina sp.
Abstract: CO2 at different concentrations were added to cultures of the eukaryotic microalgae, Chlorella kessleri, C. vulgaris and Scenedesmus obliquus, and the prokaryotic cyanobacterium, Spirulina sp., growing in flasks and in a photobioreactor. In each case, the best kinetics and carbon fixation rate were with a vertical tubular photobioreactor. Overall, Spirulina sp. had the highest rates. Spirulina sp., Sc. obliquus and C. vulgaris could grow with up to 18% CO2.
283 citations
TL;DR: The reasons for higher H2 yields during dissolved gas removal and changes in OLR will help improve H2 yield, and significant disagreement exists over the effect of organic loading rate (OLR).
Abstract: Efforts to increase H2 yields from fermentative H2 production include heat treatment of the inoculum, dissolved gas removal, and varying the organic loading rate. Although heat treatment kills methanogens and selects for spore-forming bacteria, the available evidence indicates H2 yields are not maximized compared to bromoethanesulfonate, iodopropane, or perchloric acid pre-treatments and spore-forming acetogens are not killed. Operational controls (low pH, short solids retention time) can replace heat treatment. Gas sparging increases H2 yields compared to un-sparged reactors, but no relationship exists between the sparging rate and H2 yield. Lower sparging rates may improve the H2 yield with less energy input and product dilution. The reasons why sparging improves H2 yields are unknown, but recent measurements of dissolved H2 concentrations during sparging suggest the assumption of decreased inhibition of the H2-producing enzymes is unlikely. Significant disagreement exists over the effect of organic loading rate (OLR); some studies show relatively higher OLRs improve H2 yield while others show the opposite. Discovering the reasons for higher H2 yields during dissolved gas removal and changes in OLR will help improve H2 yields.
243 citations
TL;DR: Mesorhizobium strain RC3, isolated from chickpea nodules, tolerated chromium up to 500 μg/ml and reduced it by 90% at pH 7 after 120 h and produced plant growth-promoting substances, both in the presence and absence of chromium.
Abstract: Mesorhizobium strain RC3, isolated from chickpea nodules, tolerated chromium up to 500 μg/ml and reduced it by 90% at pH 7 after 120 h. It produced plant growth-promoting substances, both in the presence and absence of chromium. Strain RC3 produced 35 μg indole acetic acid/ml in Luria Bertani broth with 100 mg tryptophan/ml, which decreased with an increase in chromium concentration. Chromium application to soil at 136 mg/kg was toxic to chickpea plants but when RC3 at 136 mg/kg was also added, it increased the dry matter accumulation, number of nodules, seed yield and grain protein by 71, 86, 36 and 16%, respectively, compared to non-inoculated plants. Nitrogen in roots and shoots were increased by 46 and 40%, respectively, at 136 mg Cr/kg. The bio-inoculant decreased the uptake of chromium by 14, 34 and 29% in roots, shoots and grains, respectively.
202 citations
TL;DR: Some of the recent advances in specific enzyme systems for bacteria and yeast cells rupture and other applications are examined and emphasis is focused in biotechnological aspects of these enzymes.
Abstract: Cell wall lytic enzymes are valuable tools for the biotechnologist, with many applications in medicine, the food industry, and agriculture, and for recovering of intracellular products from yeast or bacteria. The diversity of potential applications has conducted to the development of lytic enzyme systems with specific characteristics, suitable for satisfying the requirements of each particular application. Since the first time the lytic enzyme of excellence, lysozyme, was discovered, many investigations have contributed to the understanding of the action mechanisms and other basic aspects of these interesting enzymes. Today, recombinant production and protein engineering have improved and expanded the area of potential applications. In this review, some of the recent advances in specific enzyme systems for bacteria and yeast cells rupture and other applications are examined. Emphasis is focused in biotechnological aspects of these enzymes.
177 citations
TL;DR: A successful and marketable product would be a highly purified bacteriophage preparation containing one or several fully characterized phages, accompanied by optimized methods of administration and backed up by properly controlled efficacy and safety studies.
Abstract: The challenges for successful launching of a profitable phage therapeutic product include intellectual property rights, safety issues, reproducibility, stability and robustness of the product. A successful and marketable product would be a highly purified bacteriophage preparation containing one or several fully characterized phages, accompanied by optimized methods of administration and backed up by properly controlled efficacy and safety studies.
168 citations
TL;DR: AM1 medium was formulated with low levels of alkali metals and total salts and was equally effective for ethanol production from xylose and lactate production from glucose with average productivities of 18–19 mmol l−1 h−1 for both.
Abstract: Individual nutrient salts were experimentally varied to determine the minimum requirements for efficient L (+)-lactate production by recombinant strains of Escherichia coli B. Based on these results, AM1 medium was formulated with low levels of alkali metals (4.5 mM and total salts (4.2 g l(-1)). This medium was equally effective for ethanol production from xylose and lactate production from glucose with average productivities of 18-19 mmol l(-1) h(-1) for both (initial 48 h of fermentation).
165 citations
TL;DR: The interaction between fifteen isolates of Trichoderma harzianum and the soil-borne plant pathogen, Rhizoctonia solani, was studied and it was revealed that growth inhibition of the pathogen occurred soon after contact with the antagonist.
Abstract: The genus Trichoderma is a potential biocontrol agent against several phytopathogenic fungi. One parameter for its successful use is an efficient coiling process followed by a substantial production of hydrolytic enzymes. The interaction between fifteen isolates of Trichoderma harzianum and the soil-borne plant pathogen, Rhizoctonia solani, was studied by light microscopy and transmission electron microscopy (TEM). Macroscopic observations of fungal growth in dual cultures revealed that growth inhibition of the pathogen occurred soon after contact with the antagonist. All T. harzianum isolates tested exhibited coiling around the hyphae of R. solani. The strains ALL23, ALL40, ALL41, ALL43 and ALL49 did not differ in coiling frequency and gave equal coiling performances. No correlation between coiling frequency and the production of cell wall-degrading chitinases, N-acetyl-beta-D-glucosaminidase and beta-1,3-glucanases, was found.
164 citations
TL;DR: Artemisinin production by hairy roots of Artemisia annua L. was increased 6-fold to 1.8 μg mg−1 dry wt over 6 days by adding 150 mg chitosan l−1 by dose-dependent means.
Abstract: Artemisinin production by hairy roots of Artemisia annua L. was increased 6-fold to 1.8 μg mg−1 dry wt over 6 days by adding 150 mg chitosan l−1. The increase was dose-dependent. Similar treatment of hairy roots with methyl jasmonate (0.2 mM) or yeast extract (2 mg ml−1) increased artemisinin production to 1.5 and 0.9 μg mg−1 dry wt, respectively.
TL;DR: Though many genes of potential utility to the floricultural industry have been identified, and much has been learnt of the genetic factors and molecular mechanisms underlying phenotypes of great importance to the industry, there are only flower colour modified varieties of carnation and rose in the marketplace.
Abstract: Micro-propagation, embryo rescue, mutagenesis via chemical or irradiation means and in vitro inter-specific hybridisation methods have been used by breeders in the floriculture industry for many years. In the past 20 years these enabling technologies have been supplemented by genetic modification methods. Though many genes of potential utility to the floricultural industry have been identified, and much has been learnt of the genetic factors and molecular mechanisms underlying phenotypes of great importance to the industry, there are only flower colour modified varieties of carnation and rose in the marketplace. To a large extent this is due to unique financial barriers to market entry for genetically modified varieties of flower crops, including use of technology fees and costs of regulatory approval.
TL;DR: It is proposed that CV degradation occurs via a stepwise demethylation process to yield mono-, di-, tri-, tetra-, penta- and hexa-demethylated CV species.
Abstract: Crystal violet (CV), which has been extensively used as a biological stain and a commercial textile dye, is a recalcitrant molecule. A strain of Pseudomonas putida was isolated that effectively degraded CV: up to 80% of 60 μM CV as the sole carbon source, was degraded in liquid media within 1 week. Nine degradation products were isolated and identified. We propose that CV degradation occurs via a stepwise demethylation process to yield mono-, di-, tri-, tetra-, penta- and hexa-demethylated CV species.
TL;DR: Administration of B. licheniformis can improve the white shrimp's intestinal microflora and its immune ability.
Abstract: When Bacillus licheniformis was administered to the white shrimp, Litopenaeus vannamei, although the total bacterial counts in the intestinal tract of the shrimp remained constant, Vibrio numbers significantly decreased (P < 0.05). Haemocyte counts together with phenoloxidase and superoxide dismutase activities of the shrimp were significantly higher (P < 0.05) in treatments than in the control. Thus, administration of B. licheniformis can improve the white shrimp's intestinal microflora and its immune ability.
TL;DR: Adventitious roots of Echinacea purpurea were cultured in airlift bioreactors using Murashige and Skoog medium with 2 mg indole butyric acid l−1 and 50 g sucrose l−2 for the production of chichoric acid, chlorogenic acid and caftaric acid.
Abstract: Adventitious roots of Echinacea purpurea were cultured in airlift bioreactors (20 l, 500 l balloon-type, bubble bioreactors and 1,000 l drum-type bubble bioreactor) using Murashige and Skoog (MS) medium with 2 mg indole butyric acid l−1 and 50 g sucrose l−1 for the production of chichoric acid, chlorogenic acid and caftaric acid. In the 20 l bioreactor (containing 14 l MS medium) a maximum yield of 11 g dry biomass l−1 was achieved after 60 days. However, the amount of total phenolics (57 mg g−1 DW), flavonoids (34 mg g−1 DW) and caffeic acid derivatives (38 mg g−1 DW) were highest after 50 days. Based on these studies, pilot-scale cultures were established and 3.6 kg and 5.1 kg dry biomass were achieved in the 500 l and 1,000 l bioreactors, respectively. The accumulation of 5 mg chlorogenic acid g−1 DW, 22 mg chichoric acid g−1 DW and 4 mg caftaric acids g−1 DW were achieved with adventitious roots grown in 1,000 l bioreactors.
TL;DR: The improved Coomassie Brilliant Blue staining method is more sensitive than Blue Silver and more convenient than the Silver protocol and mass spectrometry results confirmed that it is suitable for subsequent proteomic research.
Abstract: A more sensitive and convenient Coomassie Brilliant Blue (CBB) staining method for visualizing proteins was developed. Compared with the modifications include the supplement of 10% (v/v) methanol into the fixing solution, an increase of an additional sensitization step and CBB raised from 0.1 to 0.125%. The improved method can detect proteins at nanogram level. The improved method is more sensitive than Blue Silver and more convenient than the Silver protocol. Mass spectrometry results confirmed that it is suitable for subsequent proteomic research.
TL;DR: A bacterial strain (Serratia sp.) that could transform chlorpyrifos to 3,5,6-trichloro-2-pyridinol (TCP) and a TCP-mineralizing fungal strain (Trichosporon sp.) were isolated from activated sludge by enrichment culture technique.
Abstract: A bacterial strain (Serratia sp.) that could transform chlorpyrifos to 3,5,6-trichloro-2-pyridinol (TCP) and a TCP-mineralizing fungal strain (Trichosporon sp.) were isolated from activated sludge by enrichment culture technique. The fungus could also degrade 50 mg chlorpyrifos l(-1) within 7 days. Co-cultures completely mineralized 50 mg chlorpyrifos l(-1) within 18 h at 30 degrees C and pH 8 using a total inocula of 0.15 g biomass l(-1).
TL;DR: Yeasts and filamentous fungi have gained significant interest for the production of recombinant antibodies and antibody fragments and aspects are addressed: folding, assembly and secretion of antibody related proteins, process optimization to improve productivity and quality, proteolysis, and, as a major point of interest, glycosylation.
Abstract: Yeasts and filamentous fungi have gained significant interest for the production of recombinant antibodies and antibody fragments. The opportunities and constraints of antibody (fragment) production in these hosts are highlighted as well as cell engineering strategies to overcome the constraints. Following aspects are addressed: folding, assembly and secretion of antibody related proteins, process optimization to improve productivity and quality, proteolysis, and, as a major point of interest, glycosylation.
TL;DR: Lactobacillus delbrueckii was grown on sugarcane molasses, sugar cane juice and sugar beet juice in batch fermentation at pH 6 and at 40°C and the optical purities of d-lactic acid from the feedstocks ranged from 97.2 to 98.3%.
Abstract: Lactobacillus delbrueckii was grown on sugarcane molasses, sugarcane juice and sugar beet juice in batch fermentation at pH 6 and at 40 degrees C. After 72 h, the lactic acid from 13% (w/v) sugarcane molasses (119 g total sugar l(-1)) and sugarcane juice (133 g total sugar l(-1)) was 107 g l(-1) and 120 g l(-1), respectively. With 10% (w/v) sugar beet juice (105 g total sugar l(-1)), 84 g lactic acid l(-1) was produced. The optical purities of D: -lactic acid from the feedstocks ranged from 97.2 to 98.3%.
TL;DR: The study demonstrated for the first time the usefulness of the low-value soy molasses as a combined nitrogen- and carbon-source for SL production at a reduced cost.
Abstract: A simplified medium containing only soy molasses and oleic acid as ingredients was developed for the production of sophorolipids (SLs) from Candida bombicola. We achieved a product yield of 53 ± 3 g of purified sophorolipids per liter of starting culture volume, which is 71 ± 4% of the yield obtained with growth medium that also additionally contains the costly yeast extract and urea as nitrogen source. The large majority of the SL components existed in the lactone form (87%), and the predominant component is SL containing (ω-1)-hydroxyoleic acid as the lipid moiety. The study demonstrated for the first time the usefulness of the low-value soy molasses as a combined nitrogen- and carbon-source for SL production at a reduced cost.
TL;DR: High power ultrasound (HPU) is a technology whose application has been evaluated if not exploited in several food and beverage processes but has yet to be introduced into the wine industry.
Abstract: Industrial scale food and beverage processes that utilize microorganisms are typically faced with issues related to the exclusion, suppression or elimination of spoilage organisms. Yet the use of traditional anti-microbial treatments such as heat, chemical biocides or sterile filtration may themselves be restricted by regulations or else be undesirable due to their adverse sensory impacts on the product. High power ultrasound (HPU) is a technology whose application has been evaluated if not exploited in several food and beverage processes but has yet to be introduced into the wine industry. This review examines the research findings from related industries and highlights possible applications and likely benefits of the use of HPU in winemaking.
TL;DR: The isolation and purification of a novel enzyme from Aspergillus niger that hydrolyzes this mycotoxin is reported, which is a metalloenzyme as it was inhibited at 10 mM EDTA, whereas PMSF had no effect.
Abstract: Ochratoxin A is a mycotoxin produced by several Aspergillus and some Penicillium species which may be present in food and feed products. It can be enzymatically hydrolyzed into ochratoxin α and l-β-phenylalanine, thereby decreasing its toxicity. The ochratoxin A degradation capacity of Aspergillus niger is well known and here we report the isolation and purification of a novel enzyme from A. niger that hydrolyzes this mycotoxin. A wheat germ medium supplemented with ochratoxin A was used to produce the enzyme, which was purified from culture filtrate by acetone precipitation and anion exchange chromatography. An overall purification of 2.5-fold with a recovery of 68% and a final specific activity of 36 U/mg was obtained. The enzyme is a metalloenzyme as it was inhibited at 10 mM EDTA, whereas PMSF had no effect. The ochratoxin A hydrolytic enzyme presented a Vmax of 0.44 μM/min and a Km of 0.5 mM when the reaction was carried out at pH 7.5 and 37°C.
TL;DR: Phenyllactic acid (PLA) is a novel antimicrobial compound derived from phenylalanine (Phe).
Abstract: Phenyllactic acid (PLA) is a novel antimicrobial compound derived from phenylalanine (Phe). Lactobacillus sp. SK007, having high PLA-producing ability, was isolated from Chinese traditional pickles. When 6.1 mM phenylpyruvic acid (PPA) was used to replace Phe as substrate at the same concentration, PLA production increased 14-fold and the fermentation time decreased from 72 h to 24 h with growing cells. With resting cells, however, 6.8 mM PLA could be obtained as optimal yield using the following conditions: 12 mM PPA, 55 mM glucose, pH 7.5, 35°C and 4 h.
TL;DR: The maximum transient recombinant antibody yield was 22 mg/l, the highest ever reported for a multiliter transfection in CHO, and the transiently expressed protein had the same extent of glycosylation as the same antibody produced from a stably transfected recombinant CHO cell line.
Abstract: Cell expansion, gene transfer and protein production were all executed with a single serum-free, animal protein-free commercial medium designed for suspension-adapted Chinese hamster ovary cells (CHO DG44). This is a most important process to consider for clinical production of recombinant proteins. The transfection with polyethylenimine (PEI) was shown here to be scalable using both stirred-tank bioreactors of 3- and 150-l and novel agitated cultivation vessels (50 ml ventilated centrifuge tubes and 1-l square-shaped glass bottles) that lack any instrumentation. The transient transfections spanned a range of working volumes from 2 ml to 80 l. The maximum transient recombinant antibody yield was 22 mg/l, the highest ever reported for a multiliter transfection in CHO. The transiently expressed protein had the same extent of glycosylation as the same antibody produced from a stably transfected recombinant CHO cell line.
TL;DR: 1,3-Propanediol can be produced from glycerol by Klebsiella pneumoniae under micro-aerobic conditions and this fed-batch fermentation process has been successfully scaled up to 1 m3, suitable for the production of 1, 3-PD on a large scale.
Abstract: 1,3-Propanediol (1,3-PD) can be produced from glycerol by Klebsiella pneumoniae under micro-aerobic conditions. Recently, this fed-batch fermentation process has been successfully scaled up to 1 m3. The final 1,3-PD concentration, molar yield and volumetric productivity of 72 g l−1, 57% and 2.1 g l−1 h−1, respectively, are close to those of 75 g l−1, 61%, and 2.2 g l−1 h−1 under anaerobic conditions. This process would be suitable for the production of 1,3-PD on a large scale.
TL;DR: Concentrations of Mg2+, glycine, yeast extract, biotin, acetaldehyde and peptone were optimized by a uniform design process for ethanol production by Saccharomyces cerevisiae and a predictive mathematical model was established.
Abstract: Concentrations of Mg2+, glycine, yeast extract, biotin, acetaldehyde and peptone were optimized by a uniform design process for ethanol production by Saccharomyces cerevisiae. Using non-linear step-wise regression analysis, a predictive mathematical model was established. Concentrations of Mg2+ and peptone were identified as the critical factors: 50 mM Mg2+ and 1.5% (w/v) peptone in the medium increased the final ethanol titre from 14.2% (v/v) to 17% (v/v) in 48 h.
TL;DR: Acid, alkaline and enzymatic hydrolysis of agricultural crop wastes were compared for yields of total reducing sugars with the hydrolysates being evaluated for ethanol production using a mixed culture of Zymomonas mobilis and Candida tropicalis.
Abstract: Acid, alkaline and enzymatic hydrolysis of agricultural crop wastes were compared for yields of total reducing sugars with the hydrolysates being evaluated for ethanol production using a mixed culture of Zymomonas mobilis and Candida tropicalis. Acid hydrolysis of fruit and vegetable residues gave 49-84 g reducing sugars l(-1) and 29-32 g ethanol l(-1) was then obtained. Alkaline hydrolysis did not give significant amount of reducing sugars. Enzymatic hydrolysis of fruit and vegetable residues yielded 36-123 g reducing sugars l(-1) and 11-54 g ethanol l(-1).
TL;DR: An inoculum of 1 g dry cells l−1 gave the best yield of the process, but the pH decrease affected the microorganism capacity to consume glucose and convert it into lactic acid.
Abstract: Chemically pre-treated brewer's spent grain was saccharified with cellulase producing a hydrolysate with approx. 50 g glucose l(-1). This hydrolysate was used as a fermentation medium without any nutrient supplementation by Lactobacillus delbrueckii, which produced L-lactic acid (5.4 g l(-1)) at 0.73 g g(-1) glucose consumed (73% efficiency). An inoculum of 1 g dry cells l(-1) gave the best yield of the process, but the pH decrease affected the microorganism capacity to consume glucose and convert it into lactic acid.
TL;DR: The application of five water-soluble, halogen-free, alkylammonium-based ionic liquids (ILs) as additives for advanced crystallization of lysozyme was investigated and it was revealed that the addition of some of the ILs led to less crystal polymorphism and precipitation was avoided reliably even at larger NaCl concentrations.
Abstract: The application of five water-soluble, halogen-free, alkylammonium-based ionic liquids (ILs) as additives for advanced crystallization of lysozyme was investigated. Their biocompatibility was determined by long-term measurement of the overall mean relative enzyme activities. These were maximally reduced by about 10–15% when up to 200 g IL l−1 was added. Sitting-drop vapor diffusion crystallization experiments revealed that the addition of some of the ILs led to less crystal polymorphism and precipitation was avoided reliably even at larger NaCl concentrations. The addition of ILs tended to result in larger crystals. The kinetics of lysozyme crystallization were significantly enhanced using ILs as crystallization additives, e.g. by a factor of 5.5 when 100 g ethanolammonium formate l−1 was added. ILs with “soft” anions, such as formate or glycolate, were superior to ILs with “hard” anions, like nitrate.
TL;DR: Mass spectrometry analysis suggested that disulfide bond scrambling can be prevented by specifically modifying free sulhydryl using alkylation and thus reduced the amount of artifacts on non-reducing SDS-PAGE.
Abstract: SDS-PAGE under non-reducing conditions is one of the most commonly used techniques for recombinant monoclonal antibody purity and stability indicating assay. On non-reducing SDS-PAGE, bands with a lower molecular weight than the intact antibody are routinely observed and is a common feature of IgG molecules. These fragments were analyzed by in-gel digestion followed by matrix-assisted-laser-desorption-ionization time-of-flight (MALDI-TOF) mass spectrometry, Western blot and by comparing the banding pattern of sample prepared in the presence of a reducing reagent. The fragments bands were identified as antibody lacking one light chain, two heavy chains, one light chain and one heavy chain, free heavy chain and free light chain. Sensitivity of fragmentation to sample buffer pH, incubation time, reducing reagent and alkylation reagents indicated that fragments were formed during sample preparation, but not present in the samples analyzed. Disulfide bond scrambling and β-elimination are the two major mechanisms of the formation antibody fragments. Mass spectrometry analysis suggested that disulfide bond scrambling can be prevented by specifically modifying free sulhydryl using alkylation and thus reduced the amount of artifacts on non-reducing SDS-PAGE. Breakage of disulfide bonds by β-elimination was evidenced by the detection of dehydroalanine using mass spectrometry.