scholarly journals Production Optimization of an Active β-Galactosidase of Bifidobacterium animalis in Heterologous Expression Systems

2019 ◽  
Vol 2019 ◽  
pp. 1-10 ◽  
Author(s):  
Xinxin Xu ◽  
Xiaohu Fan ◽  
Chao Fan ◽  
Xing Qin ◽  
Bo Liu ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Culture Medium ◽  
Specific Activity ◽  
Food Additives ◽  
Production Optimization ◽  
E Coli ◽  
Bifidobacterium Animalis ◽  
Wide Ph Range ◽  
Heterologous Expression Systems ◽  
Ph Range

β-Galactosidase (E.C.3.2.1.23) catalyzes the hydrolysis of lactose into glucose and galactose and the synthesis of galacto-oligosaccharides as well. The β-galactosidases from bacteria, especially lactobacilli, and yeast have neutral pH and are much more likely to be developed as food additives. However, the challenges of cumbersome purification, product toxicity, and low yield in protein production have limited the commercialization of many excellent candidates. In this study, we identified a β-galactosidase gene (bg42-106) in Bifidobacterium animalis ACCC05790 and expressed the gene product in Escherichia coli BL21(DE3) and Pichia pastoris GS115, respectively. The recombinant bG42-106 purified from E. coli cells was found to be optimally active at pH 6.0 and 60°C and had excellent stability over a wide pH range (5.0–8.0) and at high temperature (60°C). The specific activity of bG42-106 reached up to 2351 U/mg under optimal conditions. The galacto-oligosaccharide yield was 24.45 g/L after incubation with bG42-106 at 60°C for 2 h. When recombinant bG42-106 was expressed in Pichia pastoris GS115, it was found in the culture medium but only at a concentration of 1.73 U/ml. To increase its production, three strategies were employed, including codon optimization, disulfide formation, and fusion with a Cherry tag, with Cherry-tag fusion being most effective. The culture medium of P. pastoris that expressed Cherry-tagged bG42-106 contained 24.4 U/mL of β-galactosidase activity, which is 14-fold greater than that produced by culture of P. pastoris harboring wild-type bG42-106.

A novel phytase from Citrobacter gillenii: characterization and expression in Pichia pastoris (Komagataella pastoris)

2020 ◽  
Author(s):  
Artur A Tkachenko ◽  
Anna N Kalinina ◽  
Larisa N Borshchevskaya ◽  
Sergey P Sineoky ◽  
Tatiana L Gordeeva
Keyword(s):  
Amino Acid ◽  
Pichia Pastoris ◽  
Amino Acid Sequence ◽  
Specific Activity ◽  
Gene Encoding ◽  
Maximum Reaction Rate ◽  
Maximum Reaction ◽  
Wide Ph Range ◽  
Recombinant Phytase ◽  
Ph Range

Abstract The phyCg gene encoding a new phytase from C. gillenii was optimized, synthesized, cloned, and expressed in Pichia pastoris. Analysis of the amino acid sequence of the enzyme showed that it belongs to the histidine acid phosphatase family. The amino acid sequence of the PhyCg phytase has the highest homology (73.49%) with a phytase sequence from Citrobacter braakii. The main characteristics for the purified recombinant phytase were established. The optimum pH and temperature were 4.5 and 50°C, respectively. The specific activity of the enzyme was 1577 U/mg. The Michaelis constant (Km) and the maximum reaction rate (Vmax) for sodium phytate were 0,185 mM and 2185 U/mg, respectively. The enzyme showed the pH and trypsin stability and had a high activity over a wide pH range.

scholarly journals Potential Probiotic Enterococcus faecium OV3-6 and Its Bioactive Peptide as Alternative Bio-Preservation

Foods ◽  
2021 ◽  
Vol 10 (10) ◽  
pp. 2264
Author(s):  
Thiwanya Choeisoongnern ◽  
Sasithorn Sirilun ◽  
Rungaroon Waditee-Sirisattha ◽  
Komsak Pintha ◽  
Sartjin Peerajan ◽  
...  
Keyword(s):  
Enterococcus Faecium ◽  
Ribosomal Proteins ◽  
Starter Culture ◽  
Heat Stability ◽  
Fermented Food ◽  
E Coli ◽  
Active Peptide ◽  
Wide Ph Range ◽  
Small Intestinal ◽  
Ph Range

Probiotic Enterococcus faecium OV3-6 and its secreted active peptide were characterized and investigated. The strain survived in simulated gastric and small intestinal conditions at 88.16% and 94.33%, respectively. The safety assessment revealed that the strain was shown α-hemolysis and susceptible to most clinically relevant antibiotics, but intermediate sensitivity to erythromycin and kanamycin was found. It does not harbor any virulence genes except for the efaAfm gene. Both of its living cells and the cell-free supernatants (CFS) of the strain significantly reduced the adhesion of E. coli and S. Typhi on Caco-2 cells. The strain can regulate the secretion of pro and inflammatory cytokines, IL-6 and IL-12 and induce the secretion of anti-inflammatory IL-10 of the Caco-2 cell. The strain can prevent the growth of Gram-positive strains belonging to the genera Bacillus, Carnobacterium, Listeria, and Staphylococcus. It also presented the entP gene that involves the production of bacteriocin named enterocin P. The antimicrobial peptide was matched 40% with 50S ribosomal proteins L29 (7.325 kDa), as revealed by LC-MS/MS. This active peptide exhibits heat stability, is stable over a wide pH range of 2−10, and maintains its activity at −20 and 4 °C for 12 weeks of storage. Altogether, E. faecium OV3-6 thus has potential for consideration as a probiotic and bio-preservative for applied use as a fermented food starter culture and in functional food or feed industries.

scholarly journals EXPRESSION OF b-XYLOSIDASE ENCODING GENE IN PHIS/ Bacillus megaterium MS SYSTEM

2015 ◽  
Vol 2 (1) ◽  
pp. 25 ◽  
Author(s):  
Sri Sumarsih
Keyword(s):  
Culture Medium ◽  
Culture Supernatant ◽  
Specific Activity ◽  
Agar Medium ◽  
Nitrilotriacetic Acid ◽  
Protoplast Transformation ◽  
E Coli ◽  
Recombinant Cells ◽  
Relative Molecular Weight ◽  
Encoding Gene

b-Xylosidase encoding gene from G. thermoleovorans IT-08 had been expressed in the pHIS1525/ B. megaterium MS941 system. The b-xylosidase gene (xyl) was inserted into plasmid pHIS1525 and propagated in E. coli DH10b. The recombinant plasmid was transformed into B. megaterium MS941 by protoplast transformation. Transformants were selected by growing the recombinant cells on solid LB medium containing tetracycline (10 µg/ ml). The expression of the b-xylosidase gene was assayed by overlaid the recombinant B. megaterium MS941 cell with agar medium containing 0.2% ethylumbelliferyl-b-D-xyloside (MUX). This research showed that the b-xylosidase gene was succesfully sub-cloned in pHIS1525 system and expressed by the recombinant B. megaterium MS941. Theaddition of 0.5% xylose into the culture medium could increase the activity of recombinantactivity of recombinant of recombinantb-xylosidase by 2.74 fold. The recombinant B. megaterium MS941 secreted 75.56% of the expressed b-xylosidase into culture medium. The crude extract b-xylosidase showed the optimum activity at 50° C and pH 6. The recombinant b-xylosidase was purified from culture supernatant by affinity chromatographic method using agarose containing Ni-NTA (Nickel-Nitrilotriacetic acid). The pure b-xylosidase showed a specific activity of 10.06 Unit/mg protein and relative molecular weight ± 58 kDa.

scholarly journals A newly isolated yeast as an expression host for recombinant lipase

2015 ◽  
Vol 20 (2) ◽  
Author(s):  
Siti Nurbaya Oslan ◽  
Abu Bakar Salleh ◽  
Raja Noor Zaliha Raja Abd Rahman ◽  
Thean Chor Leow ◽  
Hafizah Sukamat ◽  
...  
Keyword(s):  
Specific Activity ◽  
Alcohol Oxidase ◽  
Yeast Genome ◽  
Optimum Temperature ◽  
Pichia Guilliermondii ◽  
Methanol Induction ◽  
Expression Host ◽  
Alternative Expression ◽  
Wide Ph Range ◽  
Ph Range

AbstractPichia guilliermondii strain SO isolated from spoiled orange was developed for use as an alternative expression host by using Pichia pastoris as the model of the experiment. This is the first study to report on the capability of P. guilliermondii SO as a host to express thermostable T1 lipase from Geobacillus zalihae. Alcohol oxidase and formaldehyde dehydrogenase promoters were present in the yeast genome. Interestingly, the recombinant yeast [SO/pPICZαB/T1-2 (SO2)] took only 30 h to reach optimal production with minimal methanol induction [1.5% (v/v)] in YPTM medium, as compared to P. pastoris, which took longer to reach its optimal condition. The purification yield of the His-tagged fusion lipase was 68.58%, with specific activity of 194.58 U/mg. The optimum temperature was 65°C at pH 9 in glycine-NaOH buffer, and it was stable up to 70°C in a wide pH range from pH 5 to 12. In conclusion, a newly isolated yeast from spoiled orange has been proven suitable for use as an expression host.

Expression of Aspergillus aculeatus β-mannanase in Pichia pastoris Yeast and Analysis of Industrially Important Properties of Enzyme

2019 ◽  
Vol 35 (1) ◽  
pp. 38-44
Author(s):  
M.N. Lazareva ◽  
E.I. Semenko ◽  
S.P. Sineoky
Keyword(s):  
Pichia Pastoris ◽  
Specific Activity ◽  
High Specific Activity ◽  
Yeast Cells ◽  
Aspergillus Aculeatus ◽  
Gene Encoding ◽  
Pichia Pastoris Yeast ◽  
Efficient Expression ◽  
Ph Range ◽  
First Time

β-Mannanases are enzymes for the industrial application and they can be used, in particular, in the feed industry. The most important requirements for feed enzymes are broad pH range, thermal stability and high specific activity. The efficient expression of the man1 gene encoding Aspergillus aculeatus β-1,4-mannanases in Pichia pastoris yeast cells has been obtained for the first time. The industrially valuable properties of the enzyme were confirmed. The obtained data indicate that the man1 gene from A. aculeatus is potentially useful for the construction of industrial mannanase producers on the basis of the Pichia pastoris yeast. recombinant β-mannanase, Pichia pastoris, Aspergillus aculeatus, overexpression. The work was financially supported by State project №595-00004-18 PR and used the help of the National Bioresource Center - Russian National Collection of Industrial Microorganisms NRC «Kurchatov Institute» - GosNIIgenetika (Moscow, Russia).

scholarly journals Characterization of an Alkaline Alginate Lyase with pH-Stable and Thermo-Tolerance Property

Marine Drugs ◽  
2019 ◽  
Vol 17 (5) ◽  
pp. 308 ◽  
Author(s):  
Yanan Wang ◽  
Xuehong Chen ◽  
Xiaolin Bi ◽  
Yining Ren ◽  
Qi Han ◽  
...  
Keyword(s):  
Marine Bacterium ◽  
Specific Activity ◽  
Alginate Lyase ◽  
Initial Activity ◽  
Alginate Oligosaccharides ◽  
Wide Ph Range ◽  
Encoding Gene ◽  
Ph Range ◽  
Alginate Lyases

Alginate oligosaccharides (AOS) show versatile bioactivities. Although various alginate lyases have been characterized, enzymes with special characteristics are still rare. In this study, a polysaccharide lyase family 7 (PL7) alginate lyase-encoding gene, aly08, was cloned from the marine bacterium Vibrio sp. SY01 and expressed in Escherichia coli. The purified alginate lyase Aly08, with a molecular weight of 35 kDa, showed a specific activity of 841 U/mg at its optimal pH (pH 8.35) and temperature (45 °C). Aly08 showed good pH-stability, as it remained more than 80% of its initial activity in a wide pH range (4.0–10.0). Aly08 was also a thermo-tolerant enzyme that recovered 70.8% of its initial activity following heat shock treatment for 5 min. This study also demonstrated that Aly08 is a polyG-preferred enzyme. Furthermore, Aly08 degraded alginates into disaccharides and trisaccharides in an endo-manner. Its thermo-tolerance and pH-stable properties make Aly08 a good candidate for further applications.

scholarly journals Karakterisasi Enzim Pendegradasi AHL dari Bacillus cereus INT1c dan Bacillus sp. NTT3a

2017 ◽  
Vol 3 (1) ◽  
pp. 14-20
Author(s):  
SUSI RATNANINGTYAS ◽  
IMAN RUSMANA ◽  
ALINA AKHDIYA
Keyword(s):  
Bacillus Cereus ◽  
Specific Activity ◽  
Extracellular Enzyme ◽  
Homoserine Lactone ◽  
Signal Molecules ◽  
Bacterial Cells ◽  
Bacillus Sp ◽  
Wide Ph Range ◽  
Temperature Levels ◽  
Ph Range

Some of Gram-negative bacteria perform a phenomenon called quorum sensing (QS) to activate certain phenotypes such as pathogenicity. The bacterial cells performing QS produce N-acyl homoserine lactone (AHL) as signal molecules to communicate within a population. These molecules can be degraded by the enzyme, i.e. AHL lactonase. This study aimed to characterize the activity of AHL lactonase from Bacillus cereus INT1c and Bacillus sp. NTT3a in different pH and temperature levels. Both strains produce AHL-lactonase that could be found in intracellular and extracellular extracts. The dialysis process of extracellular AHL-lactonase of INT1c significantly increased the specific activity from 5.91 to 29.96, different from an extracellular enzyme of NTT3a that slightly increased from 4.08 to 5.39. Generally dialyzedAHL-lactonase of both B. cereus INT1c and Bacillus sp. NTT3a had activity in wide pH range with better activity in acidic pH and were not stable in high temperature with the highest activity at 30-40 oC. 

scholarly journals Comparison of Different Signal Peptides for the Efficient Secretion of the Sweet-Tasting Plant Protein Brazzein in Pichia pastoris

Life ◽  
2021 ◽  
Vol 11 (1) ◽  
pp. 46
Author(s):  
Fabrice Neiers ◽  
Christine Belloir ◽  
Nicolas Poirier ◽  
Christian Naumer ◽  
Michael Krohn ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Disulfide Bonds ◽  
Large Scale ◽  
Culture Medium ◽  
Taste Receptor ◽  
Sweet Taste ◽  
West African ◽  
Signal Peptides ◽  
Sweet Taste Receptor ◽  
Ph Range

Brazzein is a small sweet-tasting protein found in the red berries of a West African evergreen shrub, Pentadiplandra brazzeana Baillon. Brazzein is highly soluble and stable over a large pH range and at high temperatures, which are characteristics that suggest its use as a natural sweetener. However, Pentadiplandra brazzeana culture is difficult at a large scale, limiting the natural source of brazzein. Heterologous expression of brazzein has been established in numerous systems, including bacteria, yeast, and transgenic plants. Brazzein requires four disulfide bonds to be active in eliciting an intense sweet taste, and the yeast Pichia pastoris appears to be one of the best options for obtaining functional brazzein in high quantities. Employing yeast secretion in the culture medium allows us to obtain fully active brazzein and facilitate purification later. To increase yeast secretion, we compared seven different signal peptides to successfully achieve brazzein secretion using the yeast P. pastoris. The brazzein proteins corresponding to these signal peptides elicited activation of the sweet taste receptor functionally expressed in a cellular assay. Among these tested signal peptides, three resulted in the secretion of brazzein at high levels.

Characterization of proteinases in different isolates of adult Haemonchus contortus

Parasitology ◽  
2004 ◽  
Vol 130 (4) ◽  
pp. 429-435 ◽  
Author(s):  
D. L. REDMOND ◽  
R. WINDHAM
Keyword(s):  
Haemonchus Contortus ◽  
Specific Activity ◽  
Molecular Size ◽  
Integral Membrane Protein ◽  
Proteinase Activity ◽  
Effective Vaccine ◽  
Wide Ph Range ◽  
Ph Range ◽  
Different Strains ◽  
High Degree

A high degree of intra- and inter-geographical variation has been demonstrated previously in the excretory/secretory proteinases released by adult Haemonchus contortus. Proteinase activity has also been associated with host-protective ‘hidden’ antigens isolated from the gut of adult H. contortus. If similar geographical strain variation also exists within the gut-associated proteinases, this will have important implications for the development of a globally effective vaccine. The proteinases active in integral-membrane protein extracts from 3 different strains of adult H. contortus were characterized on the basis of their pH optima and molecular size. Although enzyme activity was detected over a wide pH range, the majority of proteinase activity was detected at acidic pH. Differences in specific activity and size of enzymes were observed between the 3 different parasite strains at different pH values. A high degree of conservation in reactive peptides was observed when protein extracts were probed with antisera raised to the protective hidden gut-antigen complexes isolated from the Moredun strain of H. contortus, or to bacterially expressed subcomponents thereof. Therefore, despite the observed differences in membrane-bound proteinase profiles, the similarity of the immunogenic response against these hidden antigens may be sufficient to prove protective against different geographical isolates of H. contortus.

A novel NhaD-type Na+/H+ antiporter from the moderate halophile and alkaliphile Halomonas alkaliphila

2017 ◽  
Vol 63 (7) ◽  
pp. 596-607 ◽  
Author(s):  
Yanhong Wang ◽  
Na Song ◽  
Lina Yang ◽  
Heba Abdel-motaal ◽  
Rui Zhang ◽  
...  
Keyword(s):  
Membrane Vesicles ◽  
Activity Profile ◽  
Moderate Halophile ◽  
E Coli ◽  
Significant Difference ◽  
Dependent Activity ◽  
Wide Ph Range ◽  
Ph Dependent ◽  
Ph Range ◽  
Selection Of

In this study, a NhaD-type Na+/H+ antiporter gene designated Ha-nhaD was obtained by selection of genomic DNA from the moderate halophile and alkaliphile Halomonas alkaliphila in Escherichia coli KNabc lacking 3 major Na+/H+ antiporters. The presence of Ha-NhaD conferred tolerance of E. coli KNabc to NaCl up to 0.6 mol·L–1 and to LiCl up to 0.2 mol·L–1 and to an alkaline pH. pH-dependent Na+(Li+)/H+ antiport activity was detected from everted membrane vesicles prepared from E. coli KNabc/pUC-nhaD but not those of KNabc/pUC18. Ha-NhaD exhibited Na+(Li+)/H+ antiport activity over a wide pH range from 7.0 to 9.5, with the highest activity at pH 9.0. Protein sequence alignment and phylogenetic analysis revealed that Ha-NhaD is significantly different from the 7 known NhaD-type Na+/H+ antiporters, including Dw-NhaD, Dl-NhaD, Vp-NhaD, Vc-NhaD, Aa-NhaD, He-NhaD, and Ha-NhaD1. Although Ha-NhaD showed a closer phylogenetic relationship with Ha-NhaD2, a significant difference in pH-dependent activity profile exists between Ha-NhaD and Ha-NhaD2. Taken together, Ha-nhaD encodes a novel pH-dependent NhaD-type Na+/H+ antiporter.

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