Zeily Nurachman

Raw starch–degrading α‐amylase from Bacillus aquimaris MKSC 6.2: isolation and expression of the gene, bioinformatics and biochemical characterization of the recombinant enzyme

The aims were to isolate a raw starch–degrading α‐amylase gene baqA from Bacillus aquimaris MKSC 6.2, and to characterize the gene product through in silico study and its expression in Escherichia coli.

Biochemical characterization of a raw starch degrading α-amylase from the Indonesian marine bacterium Bacillus sp. ALSHL3

An Indonesian marine bacterial isolate, which belongs to genus of Bacillus sp. based on 16S rDNA analysis and was identified as Bacillus filicolonicus according to its morphology and physiology, produced a raw starch degrading α-amylase. The partially purified α-amylase using a maize starch affinity method exhibited an optimum pH and temperature of 6.0 and 60°C, respectively. The enzyme retained 72% of its activity in the presence of 1.5 M NaCl. Scanning electron micrographs showed that the α-amylase was capable of degrading starch granules of rice and maize. This α-amylase from Bacillus sp. ALSHL3 was classified as a saccharifying enzyme since its major final degradation product was glucose, maltose, and maltotriose.

Oil productivity of the tropical marine diatom Thalassiosira sp.

To understand the potential of cultivating tropical marine diatom Thalassiosira sp. to produce biofuel, biodiesel product properties and growth characteristics of Thalassiosira sp. in three different media were investigated. After medium evaluation, significant Thalassiosira sp. cell growth was observed in both Walne and enriched seawater media, but not in plain seawater medium. The microalgae grew well in alkaline condition (pH range of 8.0-8.8). The average biomass density cultured in Walne and enriched seawater media on the 6th day was 4.36 and 2.50 g L(-1), respectively. Based on ESI-IT-MS spectra, the TAGs of algal oil were identified as POP, POO, and SOO, and the FAMEs as oleic acid methyl ester. The oil productivity of Thalassiosira sp. cultured in Walne and enriched seawater media were 150 and 290 μL L(-1) d(-1), respectively. The density and kinematic viscosity of Thalassiosira sp. biodiesel were 0.857 g mL(-1) and 1.151 mm(2) s(-1).

Construction of individual, fused, and co-expressed proteins of endoglucanase and β-glucosidase for hydrolyzing sugarcane bagasse.

At least a combination of endoglucanase (EglII) and β-glucosidase (BglZ) is required for hydrolyzing crystalline cellulose. To understand the catalytic efficiency of combination enzymes for converting biomass to sugars, EglII and BglZ were constructed in the form of individual, fused as well as co-expression proteins, and their activities for hydrolyzing sugarcane bagasse were evaluated. The genes, eglII isolated from Bacillus amyloliquefaciens PSM3.1 earlier and bglZ from B. amyloliquefaciens ABBD, were expressed extracellularly in Bacillus megaterium MS941. EglII exhibited both exoglucanase and endoglucanase activities, and BglZ belonging to the glycoside hydrolase 1 family (GH 1) showed β-glucosidase activity. A combination of EglII and BglZ showed activity on substrates Avicel, CMC and sugarcane bagasse. Specifically for hydrolyzing sugarcane bagasse, fused protein (fus-EglII+BglZ), co-expression protein (coex-BglZ+EglII), and mixed-individual protein (mix-EglII+BglZ) produced cellobiose as the main product, along with a small amount of glucose. The amount of reducing sugars released from the hydrolyzing bleached sugarcane bagasse (BSB) using fus-EglII+BglZ and mix-EglII+BglZ was 2.7- and 4.2-fold higher, respectively, than steamed sugarcane bagasse (SSB), indicating the synergetic enzymes worked better on treated sugarcane bagasse. Compared with fus-EglII+BglZ and mix-EglII+BglZ, coex-BglZ+EglII released more mol reducing sugars from SSB, indicating the enzymes were potential for biomass conversion. Additionally, coex-BglZ+EglII acted on BSB 2.5-fold faster than fus-EglII+BglZ. Thus, coex-bglZ+eglII expression system was the best choice to produce enzymes for hydrolyzing sugarcane baggase.

Biochemical characterization of a glucoamylase from Saccharomycopsis fibuligera R64

Glucoamylase from the yeast Saccharomycopsis fibuligera R64 (GLL1) has successfully been purified and characterized. The molecular mass of the enzyme was 56,583 Da as determined by mass spectrometry. The purified enzyme demonstrated optimum activity in the pH range of 5.6–6.4 and at 50°C. The activity of the enzyme was inhibited by acarbose with the IC50 value of 5 μM. GLL1 shares high amino acid sequence identity with GLU1 and GLA1, which are Saccharomycopsis fibuligera glucoamylases from the strains HUT7212 and KZ, respectively. The properties of GLL1, however, resemble that of GLU1. The elucidation of the primary structure of GLL1 contributes to the explanation of this finding.

Effect of introducing a disulphide bond between the A and C domains on the activity and stability of Saccharomycopsis fibuligera R64 α-amylase.

Native enzyme and a mutant containing an extra disulphide bridge of recombinant Saccharomycopsis fibuligera R64 α-amylase, designated as Sfamy01 and Sfamy02, respectively, have successfully been overexpressed in the yeast Pichia pastoris KM71H. The purified α-amylase variants demonstrated starch hydrolysis resulting in a mixture of maltose, maltotriose, and glucose, similar to the wild type enzyme. Introduction of the disulphide bridge shifted the melting temperature (TM) from 54.5 to 56 °C and nearly tripled the enzyme half-life time at 65 °C. The two variants have similar kcat/KM values. Similarly, inhibition by acarbose was only slightly affected, with the IC50 of Sfamy02 for acarbose being 40 ± 3.4 μM, while that of Sfamy01 was 31 ± 3.9 μM. On the other hand, the IC50 of Sfamy02 for EDTA was 0.45 mM, nearly two times lower than that of Sfamy01 at 0.77 mM. These results show that the introduction of a disulphide bridge had little effect on the enzyme activity, but made the enzyme more susceptible to calcium ion extraction. Altogether, the new disulphide bridge improved the enzyme stability without affecting its activity, although minor changes in the active site environment cannot be excluded.

A new group of glycoside hydrolase family 13 α-amylases with an aberrant catalytic triad

α-Amylases are glycoside hydrolase enzymes that act on the α(1→4) glycosidic linkages in glycogen, starch, and related α-glucans, and are ubiquitously present in Nature. Most α-amylases have been classified in glycoside hydrolase family 13 with a typical (β/α)8-barrel containing two aspartic acid and one glutamic acid residue that play an essential role in catalysis. An atypical α-amylase (BmaN1) with only two of the three invariant catalytic residues present was isolated from Bacillus megaterium strain NL3, a bacterial isolate from a sea anemone of Kakaban landlocked marine lake, Derawan Island, Indonesia. In BmaN1 the third residue, the aspartic acid that acts as the transition state stabilizer, was replaced by a histidine. Three-dimensional structure modeling of the BmaN1 amino acid sequence confirmed the aberrant catalytic triad. Glucose and maltose were found as products of the action of the novel α-amylase on soluble starch, demonstrating that it is active in spite of the peculiar catalytic triad. This novel BmaN1 α-amylase is part of a group of α-amylases that all have this atypical catalytic triad, consisting of aspartic acid, glutamic acid and histidine. Phylogenetic analysis showed that this group of α-amylases comprises a new subfamily of the glycoside hydrolase family 13.

A simple way to visualize fibrinolysis in the classroom

Laboratory demonstration, as well as biochemistry lecture, has been used to complement explanation of biochemical processes. The laboratory demonstration is very useful in teaching biochemistry to students who lack background in biology. The experimental model of fibrinolysis described here presents a complex biological reaction in simplified manner, emphasizing specific terms related to enzymes, and triggers student interest in biochemistry.