Sol–gel immobilization of Alcalase from Bacillus licheniformis for application in the synthesis of C-terminal peptide amides

Corîci, Livia; Frissen, A.E.; Zoelen, Dirk-Jan van; Eggen, Ivo F.; Péter, Fráncisc; Davidescu, Corneliu Mircea; Boeriu, Carmen G.

doi:10.1016/j.molcatb.2011.08.004

Cited by 24 publications

(13 citation statements)

References 32 publications

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“…The operational stability of immobilized biocatalysts is considered as one of the most important feature that affects their applications in various bioprocesses. Generally, enzyme leakage from the carriers and enzyme inactivation represent one of the main obstacles for immobilized enzyme applications particularly in repeated batch and continuous process [ 33 ]. The reuse of NPST-AK15 protease immobilized onto Ac–HCMSS–NH 2 nanospheres was studied in several batches for substrate hydrolysis that performed at 40 °C and pH 11.…”

Section: Resultsmentioning

confidence: 99%

Enhancement of Alkaline Protease Activity and Stability via Covalent Immobilization onto Hollow Core-Mesoporous Shell Silica Nanospheres

Ibrahim

Al‐Salamah

El‐Toni

et al. 2016

IJMS

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The stability and reusability of soluble enzymes are of major concerns, which limit their industrial applications. Herein, alkaline protease from Bacillus sp. NPST-AK15 was immobilized onto hollow core-mesoporous shell silica (HCMSS) nanospheres. Subsequently, the properties of immobilized proteases were evaluated. Non-, ethane- and amino-functionalized HCMSS nanospheres were synthesized and characterized. NPST-AK15 was immobilized onto the synthesized nano-supports by physical and covalent immobilization approaches. However, protease immobilization by covalent attachment onto the activated HCMSS–NH2 nanospheres showed highest immobilization yield (75.6%) and loading capacity (88.1 μg protein/mg carrier) and was applied in the further studies. In comparison to free enzyme, the covalently immobilized protease exhibited a slight shift in the optimal pH from 10.5 to 11.0, respectively. The optimum temperature for catalytic activity of both free and immobilized enzyme was seen at 60 °C. However, while the free enzyme was completely inactivated when treated at 60 °C for 1 h the immobilized enzyme still retained 63.6% of its initial activity. The immobilized protease showed higher Vmax, kcat and kcat/Km, than soluble enzyme by 1.6-, 1.6- and 2.4-fold, respectively. In addition, the immobilized protease affinity to the substrate increased by about 1.5-fold. Furthermore, the enzyme stability in various organic solvents was significantly enhanced upon immobilization. Interestingly, the immobilized enzyme exhibited much higher stability in several commercial detergents including OMO, Tide, Ariel, Bonux and Xra by up to 5.2-fold. Finally, the immobilized protease maintained significant catalytic efficiency for twelve consecutive reaction cycles. These results suggest the effectiveness of the developed nanobiocatalyst as a candidate for detergent formulation and peptide synthesis in non-aqueous media.

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Section: Resultsmentioning

confidence: 99%

Enhancement of Alkaline Protease Activity and Stability via Covalent Immobilization onto Hollow Core-Mesoporous Shell Silica Nanospheres

Ibrahim

Al‐Salamah

El‐Toni

et al. 2016

IJMS

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“…Although Alcalase has been immobilized by sol‐gel entrapment,69 as cross‐linked enzyme crystals (CLECs)70 or as cross‐linked enzyme aggregates (CLEAs),8 these forms were considered not optimal for continuous‐flow applications. The sol‐gel Alcalase was a stable biocatalyst but with low specific activity, while Alcalase CLECs and CLEAs were not suitable to fill into columns because of their tendency to clog.…”

Section: Resultsmentioning

confidence: 99%

A Continuous‐Flow Cascade Reactor System for Subtilisin A‐ Catalyzed Dynamic Kinetic Resolution of N‐tert‐Butyloxycarbonylphenylalanine Ethyl Thioester with Benzylamine

et al. 2016

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Alcalase (Subtilisin A) was immobilized by simple hydrophobic adsorption onto various surface-grafted macroporous silicag els resulting in easy-to-prepare and stable biocatalystse nabling the efficient kinetic resolution (KR) and dynamic kinetic resolution (DKR)o fr acemic N-Boc-phenylalanine ethyl thioester with benzylamine.T he immobilized Alcalaseb iocatalysts, which retained their activity and selectivity when stored at 4 8 8Cf or more than ay ear, were testedi ne nzymatic aminolysis in batch and continuous-flow KRs resulting in (S)-N-Bocphenylalanine benzylamide in high enantiomeric purity. In KR of the racemict hioester by Alcalasecatalyzed aminolysis in ac ontinuous-flow reactor, the productivity (specific reactionr ate, r flow )a nd enantiomeric ratio (E)w ere studied in the 0-100 8 8C range.T he effect of the temperature on base-catalyzed racemization of the non-transformed (R)-thioester in ac ontinuous-flow reactor was also investigated in the 0-150 8 8Cr ange.T he continuous-mode DKR of the racemic thioester in as erialc ascade systemo fsix biocatalyst-filled columns at 50 8 8Cf or KR and five grafteds ilica gel-filled columns at 150 8 8Cf or racemization resulted in the formation of the (S)-benzylamide in 79% conversion, 8.17 g L À1 h À1 volumetric productivity and 98% ee. This is the first example of ad ynamic kinetic resolution of an amino acid derivative in continuous-flow mode using an alternating cascade of packed-bed enzyme reactors and racemization reactors kept at different temperatures.

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“…Preparation of immobilized alcalase on sol-gel matrices. The method of immobilization enzymes was adopted on entrapment enzymes on sol-gel matrices as reported by Corici et al, [15]. Alcalase 2.4L solution batch (2.4 AU/gr) was prepared for immobilization.…”

Section: B Methodsmentioning

confidence: 99%

Synthesis of Antioxidant Peptides from Melinjo (Gnetum gnemon) Seed Protein Isolated Using Sol-Gel Immobilized Alcalase

Siswoyo

Matra

Safiera

et al. 2017

International Journal on Advanced Science, Engineering and Information Technology

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The synthetic of antioxidant peptides from melinjo (Gnetum gnemon) seed protein isolated using sol-gel immobilized alcalase was studied, a commercial alcalase was physically entrapped in sol-gel matrices system. Characteristics of protein hydrolyzed were evaluated by the degree of hydrolysis obtained (DH) and the peptide profile by FPLC and SDS-PAGE. The antioxidant activity of protein hydrolyzed was determined using ABTS cation, DPPH, and superoxide radical scavenging assays. The DH values increased very quickly during the second hour followed by a slow increase up to 12 hours. Melinjo seed protein isolated was hydrolyzed about 23% of degree hydrolysis at 50°C after 2 hours of incubation. Similar results were also obtained by FPLC and SDS-PAGE, the peptide profile indicated the sol-gel immobilized alcalase was produced of peptides with low molecular weight from melinjo seed protein isolated. Immobilized alcalase preserved the initial activity over 4 cycles of repeated use in a routine reaction. The ABTS assay, the hydrolysate presented a more than 5 times greater activity than non-hydrolyzed melinjo seed protein isolated. It's confirming the potential use of sol-gel immobilized alcalase in synthetic antioxidant peptides from melinjo seed protein isolated.

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Sol–gel immobilization of Alcalase from Bacillus licheniformis for application in the synthesis of C-terminal peptide amides

Cited by 24 publications

References 32 publications

Enhancement of Alkaline Protease Activity and Stability via Covalent Immobilization onto Hollow Core-Mesoporous Shell Silica Nanospheres

Enhancement of Alkaline Protease Activity and Stability via Covalent Immobilization onto Hollow Core-Mesoporous Shell Silica Nanospheres

A Continuous‐Flow Cascade Reactor System for Subtilisin A‐ Catalyzed Dynamic Kinetic Resolution of N‐tert‐Butyloxycarbonylphenylalanine Ethyl Thioester with Benzylamine

Synthesis of Antioxidant Peptides from Melinjo (Gnetum gnemon) Seed Protein Isolated Using Sol-Gel Immobilized Alcalase

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