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.

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 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.

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. 

Purification and properties of a nonspecific β-fructofuranosidase (inulinase) from the mushroom Panaeolus papillonaceus

1987 ◽  
Vol 33 (6) ◽  
pp. 520-524 ◽  
Author(s):  
Khana Mukherjee ◽  
S. Sengupta
Keyword(s):  
Molecular Weight ◽  
Enzyme Activity ◽  
Activity Ratio ◽  
Optimum Temperature ◽  
Ph Optimum ◽  
Purification And Properties ◽  
Inulinase Activity ◽  
Wide Ph Range ◽  
Total Molecular Weight ◽  
Ph Range

A nonspecific β-fructofuranosidase (inulinase) was purified to electrophoretic homogeneity from the culture filtrate of the mushroom Panaeolus papillonaceus. The enzyme is the first purified from a basidiomycete and consists of two subunits with a total molecular weight of 116 000. It is most active on sucrose, then on raffinose, stachyose, and inulin, in decreasing order. The sucrase/inulinase activity ratio (S/I) is 5.7. Fructose was detected as the liberated sugar from raffinose, stachyose, and inulin. The enzyme is highly thermostable with an optimum temperature range of 60–65 °C and a pH optimum of 6.0. The enzyme is stable over the pH range 4–10, and is also active over a wide pH range, exhibiting 50% activity even at pH 8.5. Iodoacetate, azide, and EDTA, at 20 mM concentration, and 1% (w/v) SDS have no effect on enzyme activity, whereas Ag+ and Hg2+ at 2 mM are highly inhibitory.

Molecular characterization of an endo-type chitosanase from the fish pathogenRenibacteriumsp. QD1

2014 ◽  
Vol 94 (4) ◽  
pp. 681-686 ◽  
Author(s):  
Peichuan Xing ◽  
Dan Liu ◽  
Wen-Gong Yu ◽  
Xinzhi Lu
Keyword(s):  
Molecular Weight ◽  
Enzyme Activity ◽  
Specific Activity ◽  
Fermentation Broth ◽  
Maximal Activity ◽  
Optimum Temperature ◽  
Sds Page ◽  
Ph Range ◽  
Tlc Analysis

Renibacteriumsp. QD1, a bacteria strain capable of hydrolysing chitosan, was isolated from the homogenate of small crabs. An extracellular chitosanase, Csn-A, was purified from the QD1 fermentation broth. The enzyme was purified to homogeneity, with a yield of eight-fold, 67% recovery and a specific activity of 1575 U/mg proteins. The molecular weight of Csn-A was estimated to be 26.1 kDa by SDS-PAGE. Unlike other chitosanases, the purified Csn-A displayed maximal activity at a pH range of 5.3–6.5, and it was stable in a broad pH range of 5.0–10.0. The optimum temperature for chitosanlytic activity was 55°C. The enzyme activity was strongly stimulated by Mn2+but inhibited by Fe3+, Cu2+, Al3+, Zn2+and SDS. TLC analysis demonstrated that Csn-A hydrolysed N-deacetylated polymeric glucosamines into chito-biose and -triose in an endo-type manner. The amino acid seuquence of Csn-A showed close identity with an uncharacterized chitosanase of strain ATCC33209.

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.

scholarly journals Decolorization of textile dyes by partially purified Pleurotus ostreatus laccase

2014 ◽  
Vol 8 (3) ◽  
pp. 64-69
Author(s):  
Shamam saady Abdulredha ◽  
Abdulkareem jasim Hashim ◽  
Abduljabar Abas Ali ◽  
Batool Imran Dheeb
Keyword(s):  
Solid State ◽  
Pleurotus Ostreatus ◽  
Specific Activity ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Textile Dyes ◽  
Optimum Temperature ◽  
Optimum Conditions ◽  
Optimum Ph ◽  
Ph Range

Pleurotus ostreatus produced 2.93 U/mg of laccase in solid state fermentation (SSF) using barley bran as substrate under optimum conditions. The optimum SSF conditions were: pH 6.5; temperature, 25Cº; inoculums size 3.5 mm and moisture content, 1:1.5 w/v. Laccase was partially purified 8.29 fold with specific activity 17.5 U/mg by ion exchange chromatography after curd enzyme concentrated by dialysis against the solid sucrose. Partially purified laccase had an optimum pH of 6.5 and was stable in the pH range from 6.5 to 7.5. The optimum temperature was 45 Cº and it displayed considerable stability within the range 15 to 45 Cº with 1h incubation as well as The ability of partial purified laccase to decolorize of textile dyes showed that the blue H3R dye was completely decolorized in all concentrations within first min while yellow FG and red 3B dyes were decolorized in different percentage.

scholarly journals Characterization of a Novel L-Asparaginase from Mycobacterium gordonae with Acrylamide Mitigation Potential

Foods ◽  
2021 ◽  
Vol 10 (11) ◽  
pp. 2819
Author(s):  
Huibing Chi ◽  
Meirong Chen ◽  
Linshu Jiao ◽  
Zhaoxin Lu ◽  
Xiaomei Bie ◽  
...  
Keyword(s):  
Food Industry ◽  
Specific Activity ◽  
Lymphoblastic Leukemia ◽  
Data Bank ◽  
Clinical Settings ◽  
Potential Candidate ◽  
Structural Identity ◽  
Wide Ph Range ◽  
Ph Range

L-asparaginase (E.C.3.5.1.1) is a well-known agent that prevents the formation of acrylamide both in the food industry and against childhood acute lymphoblastic leukemia in clinical settings. The disadvantages of L-asparaginase, which restrict its industrial application, include its narrow range of pH stability and low thermostability. In this study, a novel L-asparaginase from Mycobacterium gordonae (GmASNase) was cloned and expressed in Escherichia coli BL21 (DE3). GmASNase was found to be a tetramer with a monomeric size of 32 kDa, sharing only 32% structural identity with Helicobacter pylori L-asparaginases in the Protein Data Bank database. The purified GmASNase had the highest specific activity of 486.65 IU mg−1 at pH 9.0 and 50 °C. In addition, GmASNase possessed superior properties in terms of stability at a wide pH range of 5.0–11.0 and activity at temperatures below 40 °C. Moreover, GmASNase displayed high substrate specificity towards L-asparagine with Km, kcat, and kcat/Km values of 6.025 mM, 11,864.71 min−1, and 1969.25 mM−1min−1, respectively. To evaluate its ability to mitigate acrylamide, GmASNase was used to treat potato chips prior to frying, where the acrylamide content decreased by 65.09% compared with the untreated control. These results suggest that GmASNase is a potential candidate for applications in the food industry.

Polarographic and spectrophotometric behaviour of some N-p-phenyl substituted benzamidines

1991 ◽  
Vol 56 (12) ◽  
pp. 2791-2799 ◽  
Author(s):  
Juan A. Squella ◽  
Luis J. Nuñez-Vergara ◽  
Hernan Rodríguez ◽  
Amelia Márquez ◽  
Jose M. Rodríguez-Mellado ◽  
...  
Keyword(s):  
Spectrophotometric Study ◽  
Absorption Bands ◽  
Electrochemical Parameters ◽  
Wide Ph Range ◽  
Group A ◽  
Substituent Constants ◽  
Ph Range ◽  
The Waves

Five N-p-phenyl substituted benzamidines were studied by DC and DP polarography in a wide pH range. Coulometric results show that the overall processes are four-electron reductions. Logarithmic analysis of the waves indicate that the process are irreversible. The influence of the pH on the polarographic parameters was also studied. A UV spectrophotometric study was performed in the pH range 2-13. In basic media some variations in the absorption bands were observed due to the dissociation of the amidine group. A determination of the pK values was made by deconvolution of the spectra. Correlations of both the electrochemical parameters and spectrophotometric pK values with the Hammett substituent constants were obtained.

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