Cloning and characterization of an acidic lipase from a lipolytic bacterium in tempeh.

BACKGROUND: Lipases have emerged as essential biocatalysts, having the ability to contribute to a wide range of industrial applications. Microbial lipases have garnered significant industrial attention due to their stability, selectivity, and broad substrate specificity. In the previous study, a unique lipolytic bacterium (Micrococcus luteus EMP48-D) was isolated from tempeh. It turns out the bacteria produce an acidic lipase, which is important in biodiesel production. Our main objectives were to clone the acidic lipase and investigate its potential in biodiesel production. RESULT: In this study, the gene encoding a lipase from M. luteus EMP48-D was cloned and expressed heterologously in Escherichia coli. To our knowledge, this is the first attempt at the cloning and expression of the lipase gene from Micrococcus luteus. The amino acid sequence was deduced from the nucleotide sequence (1356 bp) corresponded to a protein of 451 amino acid residues with a molecular weight of about 40 kDa. The presence of a signal peptide suggested that the protein was extracellular. A sequence analysis revealed that the protein had a lipase-specific Gly-X-Ser-X-Gly motif. The enzyme was identified as an acidic lipase with a pH preference of 5.0. Fatty acid preferences for enzyme activities were C8 and C12 (p-nitrophenyl esters), with optimum temperatures at 30-40 °C and still remaining active at 80°C. The enzyme was also shown to convert up to 70% of the substrate into fatty acid methyl ester. CONCLUSION: The enzyme was a novel acidic lipase that demonstrated both hydrolytic and transesterification reactions. It appeared particularly promising for the synthesis of biodiesel as this enzyme's catalytic reaction was optimum at low temperatures and was still active at high temperatures.

Purification and proteomic analysis of potent fibrinolytic enzymes extracted from Lumbricus rubellus.

BACKGROUND: Lumbrokinase derived from earthworms, Lumbricus rubellus is known to have fibrinolytic enzymes that have potential as therapeutic drugs due to its ability to dissolve fibrin. The current study is aimed to purify the Lumbrokinase from L. rubellus and identify its protein component. METHODS: Water extract of local earthworm Lumbricus rubellus revealed several proteins. Therefore, to identify its protein component, purification through HiPrep DEAE fast flow and proteomic analysis were conducted prior to identifications. A combination of two-dimension gel electrophoresis (2DE) and electrospray ionization mass spectrometry analysis was used to identify the purified fractions. RESULTS: The purified fractions contain five protein bands, namely F25-1, F25-2, F85-1, F85-2, and F85-3, which displayed strong fibrinogenolytic activity. F25 fractions showed fibrinogenolytic activity of 974.85 U/mg, while F85 fractions showed higher activity of 1,484.11 U/mg. Fractions F85-1, F85-2, and F85-3 showed molecular weights of 42.6 kDa, 27.03 kDa, and 14 kDa, respectively and were identified as Lumbrokinase iso-enzymes. CONCLUSION: This preliminary study indicates that the F25 and F85 fractions are similar to published fibrinolytic protease-1 and lumbrokinase, respectively, in terms of their amino acid sequence.

Effect of processing treatments on the allergenicity of nuts and legumes: A meta-analysis.

The effective food processing to reduce nuts and legumes allergenicity could not be easily and directly concluded from reading a few published reports. Therefore, we conducted a meta-analysis to investigate this issue. A literature search was conducted in eight electronic databases from January 2000 to June 11, 2021. The primary outcome of interest was the allergenicity of processed nuts or legumes determined by enzyme-linked immunosorbent assay from in vitro studies. Data with the standardized mean difference (SMD) of 95% confidence interval (CI) were pooled using a random-effect model by RevMan 5.4 software. Heterogeneity was assessed using Cochran's Q (PQ ) and I2 tests. The search strategy identified 18,793 articles. However, only 61 studies met the inclusion criteria and were included in this meta-analysis. There were 21 and 15 types of respective single and combined food processing treatments analyzed for their effects on reducing allergenicity. In single processing treatment, the extrusion and fermentation had the largest reduction in allergenicity, considering their SMD value, that is, -20.19 (95% CI: -22.22 to -18.17; the certainty of evidence: moderate) and -20.8 (95% CI: -24.10 to -17.50; the certainty of evidence: moderate), respectively. Whereas in the combination, the treatment of fermentation followed by proteolytic hydrolysis showed the most significant reduction (SMD: -53.34; 95% CI: -70.18 to -36.5) and the evidence quality of this treatment was considered moderate. In conclusion, these three food processing methods showed a desirable impact in reducing nuts or legumes allergenicity. PRACTICAL APPLICATION: Nuts and legumes play an essential role as protein sources in food consumption worldwide, but they usually contain allergens. Our study has investigated the food processing methods that effectively reduce their allergenicity by meta-analysis. The result gives valuable information for further laboratory investigation on allergens and can be used by food industries in providing foods from nuts and legumes with lower allergenicity.

Emulsifying properties and antioxidant activity of soy protein isolate conjugated with tea polyphenol extracts.

Functional properties of proteins, such as emulsification, foam formation and antioxidant activity, can be improved by conjugating the proteins with phenolic substances. We reported here changes in the structural, physicochemical and functional properties of Soy Protein Isolate (SPI) after conjugation with phenolic substances in the green tea and black tea extracts. Conjugation of SPI with tea extracts were conducted using alkaline treatment (pH 9.0) followed by air exposure. The results showed that conjugation of SPI increased the protein molecular size and decreased the protein hydrophobicity. The hydrophobic decreasing effect by the treatment was larger with the black tea extract than by green tea extract. SPI-tea polyphenol conjugates significantly (p < 0.05) increased the emulsifying ability of SPI up to 43% and the emulsifying stability of SPI up to 59%. SPI-tea polyphenol conjugates which was prepared using 0.75% (w/w SPI) green tea polyphenol extract showed the best emulsifying properties with strong repulsion forces between the droplets, smaller emulsion droplet size and lower polydispersity index of droplets size distribution. Although the conjugation product is still inferior to egg lecithin in emulsion stability, antioxidant activity of SPI was significantly (p < 0.05) improved in a concentration dependant manner. SPI-black tea polyphenol conjugates showed greater antioxidant activity than SPI-green tea polyphenol conjugate. The present study shows the feasibility and benefits of the use of SPI-tea polyphenol conjugates as a food emulsifier.

Proteomic study of bioactive peptides from tempe.

Tempe is a traditional Indonesian fermented soybean mostly produced in small industries and sold locally throughout the country. Studies on the bioactive peptides in tempe are rare. Here, we studied bioactive peptides in samples from three tempe producers with different degrees of sanitation. The peptide sub-fractions of tempe from each producer were collected following water extraction, ultrafiltration (<3 kDa), gel filtration chromatography, and reversed phase-high performance liquid chromatography (RP-HPLC) separation followed by liquid chromatography-mass spectrometry (LC-MS). The MS spectra were then predicted using FindPept tools, and their biofunctionalities were confirmed with BIOPEP databases. There were few similar peptides found in tempe from the three producers. Peptides Val-His and Ala-Leu-Glu-Pro were found in tempe from all producers. Producers having a good sanitation level had more bioactive peptides than those with moderate or poor sanitation levels (58%, 43% and 35%, from good to poor sanitation). This work showed that the tempe from the three producers had antihypertensive, antidiabetic, antioxidative and antitumor peptides.

Purification and Characterization of a Fibrinolytic Enzyme from Bacillus pumilus 2.g Isolated from Gembus, an Indonesian Fermented Food.

Bacillus pumilus 2.g isolated from gembus, an Indonesian fermented soybean cake, secretes several proteases that have strong fibrinolytic activities. A fibrinolytic enzyme with an apparent molecular weight of 20 kDa was purified from the culture supernatant of B. pumilus 2.g by sequential application of ammonium sulfate precipitation, ion-exchange chromatography, and hydrophobic chromatography. The partially purified enzyme was stable between pH 5 and pH 9 and temperature of less than 60°C. Fibrinolytic activity was increased by 5 mM MgCl2 and 5 mM CaCl2 but inhibited by 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM sodium dodecyl sulfate (SDS), and 1 mM ethylenediaminetetraacetic acid (EDTA). The partially purified enzyme quickly degraded the α and ß chains of fibrinogen but was unable to degrade the γ chain.

Fermentation RS3 derived from sago and rice starch with Clostridium butyricum BCC B2571 or Eubacterium rectale DSM 17629.

Resistant starch type 3 (RS3) is retrograded starch which is not digested by human starch degrading enzyme, and will thus undergo bacterial degradation in the colon. The main fermentation products are the Short Chain Fatty Acid (SCFA): acetate, propionate and butyrate. SCFA has significant benefit impact on the metabolism of the host. The objectives of this research were to study the SCFA profile produced by colonic butyrate producing bacteria grown in medium containing RS3. RS3 was made from sago or rice starch treated with amylase, pullulanase and the combination of amylase and pullulanase. Fermentation study was performed by using Clostridium butyricum BCC B2571 or Eubacterium rectale DSM 17629, which has been identified as capable of degradation of starch residue and also regarded as beneficial bacteria. Experimental result revealed that enzyme hydrolysis of retrograded sago or rice starch was beneficial to RS formation. RS3 derived from sago contained higher RS (31-38%) than those derived from rice starch (21-26%). This study indicated that C. butyricum BCC B2571 produced acetate, propionate and butyrate at molar ratio of 1.8 : 1 : 1, when the medium was supplemented with RSSA at concentration 1%. In the medium containing similar substrate, E. rectale DSM 17629 produced acetate, propionate and butyrate at molar ratio of 1.7 : 1 : 1.2. High levels of acetate, propionate and butyrate at molar ratio of 1.8 : 1 : 1.1 was also produced by E. rectale DSM 17629 in medium supplemented with RSSP at concentration 1%. The results showed that both bacteria responded differently to the RS3 supplementation. Such result provided insight into the possibility of designing RS3 as prebiotic with featured regarding SCFA released in the human colon with potential health implication.

Biochemical characteristics of chitosanase from the Indonesian Bacillus licheniformis MB-2.

Bacillus licheniformis MB-2, isolated from a hot spring water in Manado, Indonesia, secreted a unique chitosanase. Media consisted of 0.24% chitosan, 0.25% casiton, 1% MgSO4, 1.4% K2HPO4, 0.02% CaCl2 x 2H2O, 0.002% FeSO4 x 7H2O (w/v) was used for enzyme production. Purification of the enzyme through the hydrophobic interaction chromatography system (butyl Sepharose 4 FF) resulted in two major active fractions; the F2 fraction was shown as a single band at both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and zymogram analysis with apparent molecular mass of 75 kDa. The enzyme worked best at 70 degrees C and pH between 6.0 and 7.0. When incubated at 70, 80, and 90 degrees , the t(1/2) values were 26.56, 18.44, and 16.74 min, respectively with the k constant being at 0.026, 0.037, and 0.04/min. When heated at 90 degrees C, the enzyme retained its activity up to 8 h in the presence of 1 mM MnCl2. The enzyme's activity was unaffected by the presence of 1 M NaCl and 6 M urea but was decreased by 2 M of guanidine hydrochloride. Albeit the enzyme did not degrade colloidal and glycol chitin, it hydrolyzed glycol chitosan up to 0.8% and colloidal chitosan up to 11%. The 85% deacetylated (DDA) soluble chitosan was the most susceptible to this enzyme, followed by 90% and 100% DDA chitosan. The K(m app) values of the 85, 90, and 100% DDA soluble chitosans were found as 0.23, 0.24, and 0.58 mg/mL, whereas the Vmax values were 843, 668, and 261 U/mg, respectively. The hydrolysis products of F2 chitosanase at 24 h incubation (70 degrees C) were pentasaccharide (GlcN)5 and hexasaccharide (GlcN)6. The preliminary test showed inhibitory effect of chitooligosaccharides resulted from enzymatic degradation toward Pseudomonas aeruginosa, Salmonella typhimurium, Listeria monocytogenes, Bacillus cereus, Escherichia coli, and Staphylococcus aureus.

Biochemical characteristics of chitinase enzyme from Bacillus sp. of Kamojang Crater, Indonesia.

Chitinase and chitindeacetylase are enzymes capable of degrading chitin into chitooligomers and chitosan. The chitinases characterized and purified in this study were extracted from the acidophillic Bacillus sp. isolated from Kamojang Crater West Java Indonesia. When grown in liquid media containing colloidal chitin, the optimum chitinase activity of the acidophilic isolate was reached after 4-5 days of incubation. The optimum temperature and pH of the chitinase and chitin deacetylase were found at 37 degrees C and pH 5. When incubated at pH 5, the activity of chitin deacetylase was increased; after 3 h, the activity was 1.5 times of the control. The enzyme was stable at pH 4, after 2 h incubation, the activity was still 80% of the control. The chitinase and chitin deacetylase activities were not influenced by Mg(++) nor Ca(++), Ni(++) and Cu(++) inhibited the chitinase activity, while chitin deacetylase activity was not affected by Cu(++) addition. When 1 mM of EDTA was added, the enzyme activity was reduced 40 to 50%.