Saturation mutagenesis at Ser196 in BaCsn46A from Bacillus Amyloliquefaciens Enhances Enzyme Activity and Thermostability

The chitosanase (BaCsn46A) was extracted from Bacillus amyloliquefaciens (GenBank: QEK97559.1) and synthesized after codon optimization. The saturation mutation site was determined by analyzing the sequence and three-dimensional protein model. WT and mutant chitosanase genes were cloned and expressed in E. coli BL21 (DE3). The enzymatic properties of WT and mutants were compared, including the optimal reaction pH, temperature and thermostability. Three mutants S196F, S196Y and S196A with the highest specific enzyme activity were selected for further study. Compared with WT, the specific enzyme activity of S196Y increased by 144.76% (more than other two mutants), and the thermostability was not significantly improved. While the specific enzyme activity of S196A increased by 118.79%, and the thermostability of S196A was much higher than WT. From the perspective of industrial production, S196A is more in line with the requirements of industrial production because of its excellent thermal stability at 60°C. From the results of circular dichroism spectrum, the mutation of chitosanase at Ser196 did not change the secondary protein structure. In addition, CD analysis showed that the secondary structure of WT and mutants did not change significantly, indicating that the improvement of thermostability of S196A was not related to the secondary structure.

Magnetically Agitated Nanoparticle-Based Batch Reactors for Biocatalysis with Immobilized Aspartate Ammonia-Lyase

In this study, we investigated the influence of different modes of magnetic mixing on effective enzyme activity of aspartate ammonia-lyase from Pseudomonas fluorescens immobilized onto epoxy-functionalized magnetic nanoparticles by covalent binding (AAL-MNP). The effective specific enzyme activity of AAL-MNPs in traditional shake vial method was compared to the specific activity of the MNP-based biocatalyst in two devices designed for magnetic agitation. The first device agitated the AAL-MNPs by moving two permanent magnets at two opposite sides of a vial in x-axis direction (being perpendicular to the y-axis of the vial); the second device unsettled the MNP biocatalyst by rotating the two permanent magnets around the y-axis of the vial. In a traditional shake vial, the substrate and biocatalyst move in the same direction with the same pattern. In magnetic agitation modes, the MNPs responded differently to the external magnetic field of two permanent magnets. In the axial agitation mode, MNPs formed a moving cloud inside the vial, whereas in the rotating agitation mode, they formed a ring. Especially, the rotating agitation of the MNPs generated small fluid flow inside the vial enabling the mixing of the reaction mixture, leading to enhanced effective activity of AAL-MNPs compared to shake vial agitation.

The Efek Enzim Bromelin Buah Nanas (Ananas comosus (L.) Merr) Berbasis Sediaan Gel terhadap Lebar Intertubulus Dentin

Caries tissue cleaning can use Chemo-Mechanical Caries Removal (CMCR) based on proteolytic enzymes that catalyze peptide bonds into simpler compounds. Proteolytic enzymes can be found in mature pineapple bromelain enzymes. The aim of the study is to determine the effect of gel-based bromelain enzyme with concentrations of 8%, 10% and 12% against the width of the intertubulus dentin. The bromelain enzyme is extracted using the Lowry method. Then purified using 80% ethanol and diluting become to concentrations of 8%, 10% and 12%. Diluted bromelain enzymes were formed in gel preparations based on HPMC and applied to the study sample. The results of this study indicated a widening of the intertubulus dentin. This is indicated by the variation of the dentin intertubulus width > 2µm. This widening occurs because the bromelain enzyme can hydrolyze collagen in intertubulus dentin in the absence of alpha-l-antitrypsin. Hydrolysis causes the breaking of hydrogen bonds in the triple helix- shaped tropocollagen to turn into strands of polypeptide chains. The bromelain enzyme with a concentration 10% is more effective than 8% and 12% because it is within the maximum speed limit of the enzyme so that the enzyme is saturated by its substrate and there is a difference in specific enzyme activity in each concentration.

Recycling agro-industrial waste to produce amylase and characterizing amylase–gold nanoparticle composite

Purpose Amylase being one of the most important industrial enzymes requires large-scale production. When producing an enzyme, high productivity, high purity and low production costs need to be considered. This study focuses on comparing various agro-industrial waste substrates, for production of alpha-amylase using Bacillus amyloliquefaciens. Moreover, it studies the stability and activity of amylase–gold nanoparticles composite. Methods This study is divided into two parts, in the first part various agro-industrial waste substrates, such as wheat bran, rice bran and potato peel were used to produce alpha-amylase using solid-state fermentation (SSF). The production of the enzyme was quantified and compared in specific enzyme activity units. In the second part, change in the stability and activity of amylase in enzyme–gold nanoparticles (AuNPs) composite has been discussed. Results Highest enzyme production was observed in wheat bran and potato peel substrate with specific enzyme activity of almost 1.2 U/ug and 1.1 U/ug. Among combination substrates, wheat bran with potato peel showed a high enzyme production of 1.3 U/ug. On the other hand, the optimum temperature for amylase activity shifted to 55 °C in the composite compared to 37 °C for free enzyme. Conclusions Comparison of specific enzyme activity of extracts from various substrates showed that wheat bran alone, and in combination with potato peel, produces active and pure amylases. To stress on various catalytic activities of alpha-amylase, the capability of the enzyme to synthesize gold nanoparticles and the effect of conjugation of the nanoparticle on its optimum catalytic activity are also discussed in this paper.

The Comparative Study of Papain Enzyme from Papaya Fruits California variant and Indonesian Local variant

Papain (E.C.3.4.22.2) is a proteolytic enzyme which has important role due toits diverse uses in textile, pharmaceutics, cosmetics and food industries.Papain enzyme can be found in almost all parts of the papaya plant and mostof the stem and fruit. The objective of this study is to compare the Californiavar. and Indonesian local var. of papaya fruits, in papain production and alsoto characterize the enzyme properties. Results showed that the highest yield ofcrude papain was obtained from local papaya latex (24.87%) whichprecipitated by ethanol with ratio of 1:2. The highest of activity enzyme,soluble protein and specific enzyme activity obtained from the local papayawere 3154 ± 11.31unit/mL, solubility protein of 0.94± 0.08 mg/mL andspesific enzyme activity of 3355.32 unit/mg protein, respectively. The activityof enzyme fraction F7 obtained from purification by DEAE sepharose columnwas 202.33 U/mL dan the molecular weight of this fraction was between 17-28 kDa.© 2

Isolation and identification of bacterial protease enzyme of leather waste

The objectives of this study were to isolate and identify bacteria which produced protease enzyme from liquid and solid waste of tannery, and their characterization of enzymatic activities. The bacterial isolation used a sample of liquid and solid waste from leather waste which taken from a different waste reservoirs (three liquid waste and one solid waste in unhairing phase). Data of the isolated bacteria, OD 600 nm bacterial growth, the identification of bacteria, and enzyme precipitation with 60% ammonium sulfate were analyzed descriptively. The colony diameter, diameter of clear zone, proteolytic index, and enzymatic activities characterization on difference of pH and temperature were analyzed using Completely Randomized Design, followed by Duncan’s New Multiple Range Test. The second sample from four samples of the isolated bacteria was tested further for their proteolytic activity. The morphology of the colony was circle, white, flat ledges and convex elevation, the basal cell morphology was red, gram-negative, non-motile, catalase positive and gelatin negative. The highest activity of enzyme on pH 11 with activity unit enzyme 45,18±1,77 U/ml and specific enzyme activity 43,19±1,69 U/mg and temperature of 40°C activity unit enzyme 54,02±1,89 U/ml and specific enzyme activity 51,65±1,8 U/mg. The activity of enzyme from protease were precipitated ammonium sulfate 60% showed a higher result of (75,8 U/ml) rather than rough protease.