Isolation and Characterization of a New Ferulic-Acid-Biotransforming Bacillus megaterium from Maize Alkaline Wastewater (Nejayote).

Nejayote is an alkaline wastewater generated during the nixtamalization process. Nejayote contains high-value compounds such as ferulic acid (FA), which is widely employed as a substrate for the biotechnological production of flavors and aromas. In the present study, the isolation, identification, and characterization of a native strain of Bacillus megaterium were performed, and its capacity to produce 4-vinylguaiacol (4VG) from ferulic acid was evaluated by employing growing cell and resting cell systems. Growing cells of native B. megaterium biotransformed 6 mM crude FA in nejayote into 2.1 mM 4VG, reaching a productivity of 0.21 mM h-1 4VG, while nejayote enriched with FA at 10, 15, and 25 mM resulted in the formation of 2.4, 3.8, and 6.2 mM 4VG and productivities of 0.24, 0.38, and 0.51 mM h-1 4VG, respectively. In the resting cell system, from 6 and 25 mM pure FA, 3.5 mM 4VG was produced (0.18 mM h-1 4VG), while at 10 and 15 mM FA, 4.6 and 5.1 mM 4VG (average of 0.24 mM h-1 4VG) were obtained, respectively. The native B. megaterium strain, isolated from nejayote, showed great biotechnological potential to produce 4VG from crude FA contained in this wastewater, in which other Bacillus species, such as B. licheniformis and B. cereus, were unable to grow and biotransform FA into 4VG.

Purification and characterization of chymotrypsin from viscera of vermiculated sailfin catfish, Pterygoplichthys disjunctivus, Weber, 1991.

Pterygoplichthys disjunctivus viscera chymotrypsin was purified by fractionation with ammonium sulfate (30-70 % saturation), gel filtration, affinity, and ion exchange chromatography. Chymotrypsin molecular weight was approximately 29 kDa according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), shown a single band in zymogram. Electrofocusing study suggested being an anionic enzyme (pI ≈ 3.9), exhibiting maximal activity at pH 9 and 50 °C, using Suc-Ala-Ala-Pro-Phe-p-nitroanilide (SAAPNA) as substrate. Enzyme was effectively inhibited by phenyl methyl sulfonyl fluoride (PMSF) (99 %), and N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) (94 %). Enzyme activity was affected by the following ions in decreasing order: Hg(2+), Fe(2+), Cu(2+), Li(1+), Mg(2+), K(1+), Mn(2+), while Ca(2+) had no effect. Chymotrypsin activity decreased continuously as NaCl concentration increased (from 0 to 30 %). K m and V max values were 0.72 ± 1.4 mM and 1.15 ± 0.06 µmol/min/mg of protein, respectively (SAAPNA as substrate). Results suggest the enzyme has a potential application where low processing temperatures are needed, such as in fish sauce production.

Trypsin from viscera of vermiculated sailfin catfish, Pterygoplichthys disjunctivus, Weber, 1991: its purification and characterization.

Pterygoplichthys disjunctivus viscera trypsin was purified by fractionation with ammonium sulphate, gel filtration, affinity and ion exchange chromatography (DEAE-Sepharose). Trypsin molecular weight was approximately 27.5kDa according to SDS-PAGE, shown a single band in zymography. It exhibited maximal activity at pH 9.5 and 40°C, using N-benzoyl-dl-arginine-p-nitroanilide (BAPNA) as substrate. Enzyme was effectively inhibited by phenyl methyl sulphonyl fluoride (PMSF) (100%), N-α-p-tosyl-l-lysine chloromethyl ketone (TLCK) (85.4%), benzamidine (80.2%), and soybean trypsin inhibitor (75.6%) and partially inhibited by N-tosyl-l-phenylalanine chloromethyl ketone (TPCK) (10.3%), ethylendiaminetetraacetic acid (EDTA) (8.7%) and pepstatin A (1.2%). Enzyme activity was slightly affected by metal ions (Fe(2+)>Hg(2+)>Mn(2+)>K(+)>Mg(2+)>Li(+)>Cu(2+)). Trypsin activity decreased continuously as NaCl concentration increased (0-30%). Km and kcat values were 0.13mM and 1.46s(-1), respectively. Results suggest the enzyme have a potential application where room processing temperatures (25-35°C) or high salt (30%) concentration are needed, such as in fish sauce production.

Acidic proteases from Monterey sardine (Sardinops sagax caerulea) immobilized on shrimp waste chitin and chitosan supports: searching for a by-product catalytic system.

Solid wastes generated from the seafood industry represent an important environmental pollutant; therefore, utilization of those wastes for the development of processing biochemical tools could be an attractive and clean solution for the seafood industry. This study reports the immobilization of semi-purified acidic proteases from Monterey sardine stomachs onto chitin and chitosan materials extracted from shrimp head waste. Several supports (chitosan beads, chitosan flakes, and partially deacetylated flakes) were activated either with genipin or Na-tripolyphosphate and evaluated as a mean to immobilize acidic proteases. The protein load varied within the 67-91% range on different supports. The immobilization systems based on chitosan beads achieved the highest protein loads but showed the lowest retained catalytic activities. The best catalytic behavior was obtained using partially deacetylated chitin flakes activated either with genipin or Na-tripolyphosphate. According to results, the immobilization matrix structure, as well as acetylation degree of chitin-chitosan used, has considerable influence on the catalytic behavior of immobilized proteases. Partially deacetylated chitin flakes represent a suitable option as support for enzyme immobilization because its preparation requires fewer steps than other supports. Two abundant seafood by-products were used to obtain a catalytic system with enough proteolytic activity to be considered for biotechnological applications in diverse fields.