Stabilization of invertase by molecular engineering

Tananchai, Pattamawadee; Chisti, Yusuf

doi:10.1002/btpr.314

Cited by 10 publications

(8 citation statements)

References 30 publications

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“…Also thermostable enzymes can improve productivity of biotechnological processes because the rate of a reaction typically doubles with every 10 °C increase in temperature (Tananchai and Chisti 2010). It is an important objective in industrial criteria which was obtained in this study and the recombinant expressed invertase has the ability to withstand and remain its activity at high temperature and can be a valuable candidate for industrial applications.…”

Section: Resultsmentioning

confidence: 99%

Cloning and expression of Saccharomyces cerevisiae SUC2 gene in yeast platform and characterization of recombinant enzyme biochemical properties

et al. 2016

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Invertase (EC.3.2.1.26) catalyzes the hydrolysis of sucrose to an equimolar mixture of d-glucose and d-fructose which is of interest for various industrial applications. In this research, Saccharomyces cerevisiae invertase gene (SUC2) was optimized based on Pichia pastoris codon preference. The synthetic gene was introduced into the methylotrophic yeast Pichia pastoris under the control of the inducible AOX1 promoter. High level of the extracellular recombinant invertase (R-inv) production was achieved via methanol induction for 4 days and purified by His-Tag affinity chromatography which appeared to be a mixture of glycosylated proteins with various sizes of 85–95 kDa on SDS-PAGE. Deglycosylation of the proteins by Endo-H resulted in the proteins with average molecular weight of 60 kDa. The purified recombinant invertase biochemical properties and kinetic parameters determined a pH and temperature optimum at 4.8 and 60 °C, respectively, which in comparison with native S. cerevisiae invertase, thermal stability of recombinant invertase is highly increased in different heating treatment experiments. The purification of recombinant invertase resulted in an enzyme with specific activity of 178.56 U/mg with 3.83-fold of purification and the kinetic constants for enzyme were Km value of 19 mM and Vmax value of 300 μmol min−1 mg−1 With kinetic efficiency (Kcat/Km) of 13.15 s−1 mmol−1 it can be concluded that recombinant P. pastoris invertase can be more effective for industrial quality criteria. We conclude that recombinant P. pastoris enzyme with broad pH stability, substrate specificity and proper thermal stability can fulfil a series of predefined industrial quality criteria to be used in food, pharmaceutical and bio ethanol production industries.

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Section: Resultsmentioning

confidence: 99%

Cloning and expression of Saccharomyces cerevisiae SUC2 gene in yeast platform and characterization of recombinant enzyme biochemical properties

et al. 2016

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“…Although its closest characterized relative, the intracellular SucB of A. niger , possesses a similar pH optima profile, its temperature optima was only recorded at 37-40°C [ 16 ]. A characteristic that CmINV shares with Suc2 is its weak thermostability after prolonged exposure to higher-than-ambient temperatures [ 23 ]. A more thermostable version of an enzyme is highly desirable as it would increase the productivity of enzyme conversion [ 24 ].…”

Section: Discussionmentioning

confidence: 99%

Identification of the gene for β-fructofuranosidase from Ceratocystis moniliformis CMW 10134 and characterization of the enzyme expressed in Saccharomyces cerevisiae

et al. 2013

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Backgroundβ-Fructofuranosidases (or invertases) catalyse the commercially-important biotransformation of sucrose into short-chain fructooligosaccharides with wide-scale application as a prebiotic in the functional foods and pharmaceutical industries.ResultsWe identified a β-fructofuranosidase gene (CmINV) from a Ceratocystis moniliformis genome sequence using protein homology and phylogenetic analysis. The predicted 615 amino acid protein, CmINV, grouped with an existing clade within the glycoside hydrolase (GH) family 32 and showed typical conserved motifs of this enzyme family. Heterologous expression of the CmINV gene in Saccharomyces cerevisiae BY4742∆suc2 provided further evidence that CmINV indeed functions as a β-fructofuranosidase. Firstly, expression of the CmINV gene complemented the inability of the ∆suc2 deletion mutant strain of S. cerevisiae to grow on sucrose as sole carbohydrate source. Secondly, the recombinant protein was capable of producing short-chain fructooligosaccharides (scFOS) when incubated in the presence of 10% sucrose. Purified deglycosylated CmINV protein showed a molecular weight of ca. 66 kDa and a Km and Vmax on sucrose of 7.50 mM and 986 μmol/min/mg protein, respectively. Its optimal pH and temperature conditions were determined to be 6.0 and 62.5°C, respectively. The addition of 50 mM LiCl led to a 186% increase in CmINV activity. Another striking feature was the relatively high volumetric production of this protein in S. cerevisiae as one mL of supernatant was calculated to contain 197 ± 6 International Units of enzyme.ConclusionThe properties of the CmINV enzyme make it an attractive alternative to other invertases being used in industry.

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“…Whereas too many amino groups on the immobilization matrix mean that a protease molecule can bind to multiple points on the matrix. Binding of the protease at too many points may reduce access of the substrate to the active site of the enzyme, or leave the enzyme molecule too rigid to be active …”

Section: Resultsmentioning

confidence: 99%

“…Too high a temperature would denature the enzyme and too low a temperature will slow the deproteinization reaction. Immobilization of the enzyme is likely to enhance its temperature stability and, therefore, the optimal deproteinization temperature using the immobilized enzyme may be different compared with the optimal temperature for the dissolved form of the same enzyme. Activity and longevity of immobilized enzyme preparations are known to be temperature dependent …”

Section: Resultsmentioning

confidence: 99%

Optimal conditions for deproteinizing natural rubber using immobilized alkaline protease

Junoi

Chisti

Hansupalak

2014

J. Chem. Technol. Biotechnol.

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BACKGROUND: Natural rubber latex contains allergenic proteins. Therefore, processes for deproteinizing the latex are needed. An immobilized enzyme process for deproteinization is reported. An optimal protocol was first developed for immobilizing a protease on cellulose−chitosan composite beads. The beads were then used in developing an optimal deproteinization treatment. The main effects and the interactions of the factors for the two processes were identified using a two-level full factorial experimental design.RESULTS: Under optimal conditions (15% cellulose in beads, immobilizing reaction pH of 9, immobilization period of 24 h), the beads attained the highest specific activity of 1685.3 U g −1 beads. Using these beads, the optimal conditions for deproteinization of the latex were: an enzyme loading of 0.1 parts per hundred of rubber (phr), a sodium dodecyl sulfate concentration of 20 phr, 30 • C, and a treatment period of 12 h. The nitrogen content of the rubber was reduced to 0.012% from an initial value of 0.3%. The enzyme beads were operationally stable and could be reused for at least five cycles. CONCLUSIONS:The optimized treatment effectively deproteinized the natural rubber. The final product was free of peptides. This deproteinization treatment was more effective than the conventional urea-based treatment performed for comparison.

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Stabilization of invertase by molecular engineering

Cited by 10 publications

References 30 publications

Cloning and expression of Saccharomyces cerevisiae SUC2 gene in yeast platform and characterization of recombinant enzyme biochemical properties

Cloning and expression of Saccharomyces cerevisiae SUC2 gene in yeast platform and characterization of recombinant enzyme biochemical properties

Identification of the gene for β-fructofuranosidase from Ceratocystis moniliformis CMW 10134 and characterization of the enzyme expressed in Saccharomyces cerevisiae

Optimal conditions for deproteinizing natural rubber using immobilized alkaline protease

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