scholarly journals High-level expression of improved thermo-stable alkaline xylanase variant in Pichia Pastoris through codon optimization, multiple gene insertion and high-density fermentation

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Yihong Lu ◽  
Cheng Fang ◽  
Qinhong Wang ◽  
Yuling Zhou ◽  
Guimin Zhang ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Pichia Pastoris ◽  
Paper Industry ◽  
Xylanase Activity ◽  
Multiple Gene ◽  
E Coli ◽  
Gene Insertion ◽  
Alkaline Conditions ◽  
Pulp Properties ◽  
High Level

Abstract In paper industry, xylanases are used to increase the pulp properties in bleaching process as its eco-friendly nature. The xylanases activity is hindered by high temperature and alkaline conditions with high enzyme production cost in the paper industry. Here, XynHB, an alkaline stable xylanase from Bacillus pumilus HBP8 was mutated at N188A to XynHBN188A. Expressed mutant in E. coli showed 1.5-fold higher xylanase activity than XynHB at 60 °C. The mutant expressed in Pichia pastoris was glycosylated, remained stable for 30 min at 60 °C. XynHBN188A optimized based on codon usage bias for P. pastoris (xynHBN188As) showed an increase of 39.5% enzyme activity. The strain Y16 forming the largest hydrolysis halo in the xylan plate was used in shake flask experiments produced an enzyme activity of 6,403 U/ml. The Y16 strain had 9 copies of the recombinant xynHBN188As gene in the genome revealed by qPCR. The enzymatic activity increased to 48,241 U/ml in a 5 L fermentor. Supplement of 15 U/g xylanase enhanced the brightness of paper products by 2% in bleaching experiment, and thereby improved the tensile strength and burst factor by 13% and 6.5%, respectively. XynHBN188As has a great potential in paper industries.

Production of mouse epidermal growth factor in yeast: high-level secretion using Pichia pastoris strains containing multiple gene copies

Gene ◽  
1991 ◽  
Vol 105 (2) ◽  
pp. 205-212 ◽  
Author(s):  
Jeffrey J. Clare ◽  
Michael A. Romanes ◽  
Frederick B. Rayment ◽  
James E. Rowedder ◽  
Marjorie A. Smith ◽  
...  
Keyword(s):  
Growth Factor ◽  
Pichia Pastoris ◽  
Epidermal Growth Factor ◽  
Multiple Gene ◽  
Gene Copies ◽  
Epidermal Growth ◽  
High Level

scholarly journals Optimization of β-D-galactosidase rapid enzyme assay using Escherichia coli ATCC 8739

2016 ◽  
Vol 14 (2) ◽  
pp. 361-367
Author(s):  
Le Thi Anh Tu ◽  
Le Ba Le
Keyword(s):  
Escherichia Coli ◽  
Enzyme Activity ◽  
E Coli ◽  
Bacterial Enzyme ◽  
Rapid Enumeration ◽  
Effects Of Ph ◽  
Plate Counts ◽  
High Level ◽  
Hydrolysis Of ◽  
Substrate Concentrations

The bacterial enzyme, β-D-galactosidase, catalyzes the breakdown of the complex sugar lactose into its components - galactose and glucose-  simple sugars. The glycolysis of lactose by β-D-galactosidase is a point of attack for studies of the biochemical problem in disaccharide utilization and the genetic basis of enzyme constitution β-D galactosidase. The aim of the present study was to focus on optimization of a rapid enumeration method based on the enzymatic hydrolysis of 4-methylumbelliferyl-β-D-galactoside (MUGal) for toxicity test using Escherichia coli (E. coli) ATCC 8739 as a model. This rapid assay is based on the assumption that β-D-galactosidase is one marker for E. coli ATCC 8739. The enzymatic activity of E. coli ATCC 8739 was measured in a 25-minute assay. The effects of pH, temperature, nutrition, and substrate concentrations on enzyme activity were investigated. The enzyme β-D-galactosidase was shown to be induced with the inducer isopropyl-beta-D-thiogalactopyranoside (IPTG). The high level of production of β-D-galactosidase was found at bacteria incubated in tryptic soy broth without dextrose with IPTG. The optimum pH and temperature for β-D-galactosidase activity of E. coli ATCC 8739 was found to be 6.8 and 44.5 oC, respectively. The enzyme activity detected increased when raising the concentrations of the substrate (MUGal). The good linear correlation between logarithms of enzyme activities and CFU showed following supported the use of this method for toxicity experiments. Enzymatic methods and reference plate counts were significantly correlated. This method is sensitive, simple to perform and can be used as an alternative for traditional methods in detecting cell viability.

scholarly journals Cloning and expression of xylanase variants in Pichia pastoris

2017 ◽  
Author(s):  
◽  
Natasha Govindarajulu
Keyword(s):  
Pichia Pastoris ◽  
Animal Feed ◽  
Negative Impact ◽  
Paper Industry ◽  
Xylanase Activity ◽  
Restriction Enzymes ◽  
Cloning And Expression ◽  
Thermomyces Lanuginosus ◽  
High Yield ◽  
Heterologous Proteins

Microbial xylanases have attracted considerable research interest because of their various applications in biotechnology including the biobleaching of kraft pulp, to increase the nutritional value of foods and animal feed as well as for their potential use in the production of ethanol and methane. In the paper and pulp industry, the bleaching process involves the use of toxic chemicals and in the interim produces harmful gases that have a negative impact on the environment. The application of enzymes for this process will potentially reduce the environmental pollution by this industry. In addition, using an enzyme that is thermostable and alkali tolerant means that they will remain active under the required processing conditions. The xylanase gene, xynA derived from Thermomyces lanuginosus DSM 5826, was previously evolved to produce a number of xylanase variants, which were further enhanced for increased thermostability and alkalinity. In this study, these variants were cloned in Pichia pastoris using the pBGP1 vector to achieve extracellular production of the recombinant proteins. The xylanase genes were isolated using PCR. Both vector and DNA inserts were linearized with restriction enzymes EcoRI and XbaI and ligated. Electroporation was employed to transform the yeast with the recombinant plasmids. This was followed by the expression of the enzymes in P. pastoris grown in yeast peptone glucose (YPD) medium. Enzyme activity was thereafter assessed and the yeast was found to produce 164, 78, 96 and 142 IU/ml of S325, S340, G41 and G53 xylanase respectively, higher levels than bacterial hosts. The enzymes were then characterized and it was established that the optimum temperatures and pH for maximum xylanase activity were, 60°C, pH 6 for S325; 40°C, pH 5 for S340; 60°C, pH 6 for G41 and 60°C, pH 7 for G53. i The pH and temperature stabilities of the respective enzymes were investigated, the S325 variant was exceptionally stable at a pH between 5 and 7 and temperature range of 40-80°C and retained a minimum of 40% of activity at higher pH and temperature after an incubation period of 90 min. The S340 variant was the least thermostable and alkali stable from all four variants, it however retained 40% of activity when subjected to conditions of pH 9, 80°C after 90 min. The G41 and G53 were highly stable under the pH and temperature conditions that they were subjected to. Thus being suitable for potential application in the pulp and paper industry. The enzymes were able to retain 80% of activity at pH 9, 80°C after 120 min. P. pastoris has been proven to be a more suitable protein expression vector than E. coli for a number of reasons, including; the ability to perform complex post-translational modifications and grow to high densities in minimal media resulting in the production of a high yield of heterologous proteins.

scholarly journals High-Level Heterologous Expression of Endo-1,4-β-Xylanase from Penicillium citrinum in Pichia pastoris X-33 Directed through Codon Optimization and Optimized Expression

Molecules ◽  
2019 ◽  
Vol 24 (19) ◽  
pp. 3515 ◽  
Author(s):  
Chanika Ouephanit ◽  
Nassapat Boonvitthya ◽  
Sophie Bozonnet ◽  
Warawut Chulalaksananukul
Keyword(s):  
Pichia Pastoris ◽  
Xylanase Activity ◽  
Alcohol Oxidase ◽  
Maximum Activity ◽  
Exchange Chromatography ◽  
Gene Encoding ◽  
Original Activity ◽  
Native Strain ◽  
Wide Range ◽  
High Level

Most common industrial xylanases are produced from filamentous fungi. In this study, the codon-optimized xynA gene encoding xylanase A from the fungus Penicilium citrinum was successfully synthesized and expressed in the yeast Pichia pastoris. The levels of secreted enzyme activity under the control of glyceraldehyde-3-phosphate dehydrogenase (PGAP) and alcohol oxidase 1 (PAOX1) promoters were compared. The Pc Xyn11A was produced as a soluble protein and the total xylanase activity under the control of PGAP and PAOX1 was 34- and 193-fold, respectively, higher than that produced by the native strain of P. citrinum. The Pc Xyn11A produced under the control of the PAOX1 reached a maximum activity of 676 U/mL when induced with 1% (v/v) methanol every 24 h for 5 days. The xylanase was purified by ion exchange chromatography and then characterized. The enzyme was optimally active at 55 °C and pH 5.0 but stable over a broad pH range (3.0–9.0), retaining more than 80% of the original activity after 24 h or after pre-incubation at 40 °C for 1 h. With birchwood xylan as a substrate, Pc Xyn11A showed a Km(app) of 2.8 mg/mL, and a kcat of 243 s−1. The high level of secretion of Pc Xyn11A and its stability over a wide range of pH and moderate temperatures could make it useful for a variety of biotechnological applications.

Does the kappa number method accurately reflect lignin content in nonwood pulps?

TAPPI Journal ◽  
2018 ◽  
Vol 17 (11) ◽  
pp. 611-617
Author(s):  
Sabrina Burkhardt
Keyword(s):  
Lignin Content ◽  
Paper Industry ◽  
Pulp And Paper Industry ◽  
Original Data ◽  
Kappa Number ◽  
Klason Lignin ◽  
Organic Functional Groups ◽  
Pulp Properties ◽  
Wood Pulps ◽  
Hexenuronic Acid

The traditional kappa number method was developed in 1960 as a way to more quickly determine the level of lignin remaining in a completed or in-progress pulp. A significantly faster approach than the Klason lignin procedure, the kappa number method is based on the reaction of a strong oxidizing agent (KMnO4) with lignin and small amounts of other organic functional groups present in the pulp, such as hexenuronic acid. While the usefulness of the kappa number for providing information about bleaching requirements and pulp properties has arguably transformed the pulp and paper industry, it has been mostly developed for kraft, sulfite, and soda wood pulps. Nonwood species have a different chemical makeup than hardwood or softwood sources. These chemical differ-ences can influence kappa and Klason measurements on the pulp and lead to wide ranges of error. Both original data from Sustainable Fiber Technologies’ sulfur and chlorine-free pulping process and kappa and Klason data from various nonwood pulp literature sources will be presented to challenge the assumption that the kappa number accurately represents lignin content in nonwood pulps.

Toxic Effect of 2-ethyl (bicyclo[2.2.1] heptane) on Bacterial Cells

2019 ◽  
Vol 35 (6) ◽  
pp. 67-72 ◽  
Author(s):  
I.V. Manukhov ◽  
L.S. Yaguzhinsky ◽  
M.V. Bermeshev ◽  
M.A. Zisman ◽  
V.G. Pevgov ◽  
...  
Keyword(s):  
Toxic Effect ◽  
Atmospheric Oxygen ◽  
Inducible Promoter ◽  
Oxygen Species ◽  
Bacterial Cells ◽  
Unique Identifier ◽  
E Coli ◽  
Lux Biosensors ◽  
High Level ◽  
Ministry Of Higher Education

Toxic effect of 2-ethylnorbornane (2-ethyl(bicyclo[2.2.1]heptane) (EBH)) on bacteria has been studied using the E. coli pRecA-lux and E. coli pKatG- lux cells as lux-biosensors. It was shown that the addition of EBH to the incubation medium leads to death and growth retardation, high level oxidative stress and DNA damage in E. coli cells. It is assumed that the oxidation of EBH with atmospheric oxygen causes the formation of reactive oxygen species in the medium, which makes a major contribution to the toxicity of this substance. biosensor, luciferase, bioluminescence, inducible promoter, PrecA, PkatG The authors are grateful to Stanislav Filippovich Chalkin for the development of interdisciplinary ties in the scientific community. The work was financially supported by the Ministry of Higher Education and Science of Russia (Project Unique Identifier RFMEFI60417X0181, Agreement No. 14.604.21.0181 of 26.09.2017).

High-Level Secretory Expression and Purification of Recombinant Human Interleukin 1 Beta in Pichia pastoris

2016 ◽  
Vol 23 (8) ◽  
pp. 763-769 ◽  
Author(s):  
Pengfei Li ◽  
Ganggang Yang ◽  
Xiaofang Geng ◽  
Jinbao Shi ◽  
Bin Li ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Interleukin 1 ◽  
Interleukin 1 Beta ◽  
Expression And Purification ◽  
Secretory Expression ◽  
High Level

scholarly journals Antibiotic tolerance is associated with a broad and complex transcriptional response in E. coli

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Heather S. Deter ◽  
Tahmina Hossain ◽  
Nicholas C. Butzin
Keyword(s):  
Transcriptional Regulatory Network ◽  
Transcriptional Response ◽  
Regulatory Proteins ◽  
Health Concern ◽  
Transcriptional Networks ◽  
Antibiotic Tolerance ◽  
Multiple Gene ◽  
E Coli ◽  
Transcriptional Regulatory ◽  
Antibiotic Efficacy

AbstractAntibiotic treatment kills a large portion of a population, while a small, tolerant subpopulation survives. Tolerant bacteria disrupt antibiotic efficacy and increase the likelihood that a population gains antibiotic resistance, a growing health concern. We examined how E. coli transcriptional networks changed in response to lethal ampicillin concentrations. We are the first to apply transcriptional regulatory network (TRN) analysis to antibiotic tolerance by leveraging existing knowledge and our transcriptional data. TRN analysis shows that gene expression changes specific to ampicillin treatment are likely caused by specific sigma and transcription factors typically regulated by proteolysis. These results demonstrate that to survive lethal concentration of ampicillin specific regulatory proteins change activity and cause a coordinated transcriptional response that leverages multiple gene systems.

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