Optimization and scale-up of α-amylase production by Aspergillus oryzae using solid-state fermentation of edible oil cakes.

BACKGROUND: Amylases produced by fungi during solid-state fermentation are the most widely used commercial enzymes to meet the ever-increasing demands of the global enzyme market. The use of low-cost substrates to curtail the production cost and reuse solid wastes are seen as viable options for the commercial production of many enzymes. Applications of α-amylases in food, feed, and industrial sectors have increased over the years. Additionally, the demand for processed and ready-to-eat food has increased because of the rapid growth of food-processing industries in developing economies. These factors significantly contribute to the global enzyme market. It is estimated that by the end of 2024, the global α-amylase market would reach USD 320.1 million (Grand View Research Inc., 2016). We produced α-amylase using Aspergillus oryzae and low-cost substrates obtained from edible oil cake, such as groundnut oil cake (GOC), coconut oil cake (COC), sesame oil cake (SOC) by solid-state fermentation. We cultivated the fungus using these nutrient-rich substrates to produce the enzyme. The enzyme was extracted, partially purified, and tested for pH and temperature stability. The effect of pH, incubation period and temperature on α-amylase production using A. oryzae was optimized. Box-Behnken design (BBD) of response surface methodology (RSM) was used to optimize and determine the effects of all process parameters on α-amylase production. The overall cost economics of α-amylase production using a pilot-scale fermenter was also studied. RESULTS: The substrate optimization for α-amylase production by the Box-Behnken design of RSM showed GOC as the most suitable substrate for A. oryzae, as evident from its maximum α-amylase production of 9868.12 U/gds. Further optimization of process parameters showed that the initial moisture content of 64%, pH of 4.5, incubation period of 108 h, and temperature of 32.5 °C are optimum conditions for α-amylase production. The production increased by 11.4% (10,994.74 U/gds) by up-scaling and using optimized conditions in a pilot-scale fermenter. The partially purified α-amylase exhibited maximum stability at a pH of 6.0 and a temperature of 55 °C. The overall cost economic studies showed that the partially purified α-amylase could be produced at the rate of Rs. 622/L. CONCLUSIONS: The process parameters for enhanced α-amylase secretion were analyzed using 3D contour plots by RSM, which showed that contour lines were more oriented toward incubation temperature and pH, having a significant effect (p < 0.05) on the α-amylase activity. The optimized parameters were subsequently employed in a 600 L-pilot-scale fermenter for the α-amylase production. The substrates were rich in nutrients, and supplementation of nutrients was not required. Thus, we have suggested an economically viable process of α-amylase production using a pilot-scale fermenter.

An iodine catalyzed metal free domino process for the stereoselective synthesis of oxygen bridged bicyclic ethers.

A domino reaction has been developed for the synthesis of oxygen bridged bicyclic ethers through the coupling of 4-(2-hydroxyethyl)cyclohex-3-enols with aldehydes in the presence of 10 mol% of molecular iodine in dichloromethane at 25 °C. This method is highly diastereoselective affording the corresponding bicyclic ethers, i.e. octahydro-4a,7-epoxyisochromenes in good yields with high selectivity. It is the first report on the synthesis of oxygen bridged bicyclic ethers using a domino Prins strategy.

Reverse turn induced pi-facial selectivity during polyaniline-supported cobalt(II) salen catalyzed aerobic epoxidation of N-cinnamoyl L-proline derived peptides.

A novel chemo- and diastereoselective aerobic epoxidation of the N-cinnamoyl peptides catalyzed by polyaniline-supported cobalt(II) salen (PASCOS) is described. The N-cinnamoyl proline derived peptides 1 show a high pi-facial selectivity during these epoxidations. The origin of this diastereoselectivity in 1 has been attributed to (i) the propensity of the N-cinnamoyl proline amide to exist predominantly as trans rotamer in CDCl3, DMSO-d6, and CH3CN medium and (ii) existence of these peptides as organized structures (gamma- and beta-turns) due to the presence of intramolecular hydrogen bonds. An extensive solution NMR and MD simulation study on 1d and 1f indicates that the origin of the high pi-facial selectivity is due to the well-defined gamma- and beta-turns which result in the hindrance of one face of the cinnamoyl double bond in the transition state of the epoxidation reaction.

Nucleation of beta-hairpin structures with cis amide bonds in E-vinylogous proline-containing peptides.

Synthesis and conformational studies of peptides containing the E-vinylogous prolines 1 (VPro1) and 2 (VPro2), Boc-Ala-Val-VPro1-Xaa-Leu-OMe (3, Xaa = Gly; 4, Xaa = Phe), Boc-Ala-Val-VPro2-Xaa-Leu-OMe (5, Xaa = Gly; 6, Xaa = Phe), Boc-Leu-Ile-Val-VPro1-Xaa-Leu-OMe (7, Xaa = Gly; 8, Xaa = Phe), and Boc-Leu-Ile-Val-VPro2-Xaa-Leu-OMe (9, Xaa = Gly; 10, Xaa = Phe), were carried out. It has been shown that both VPro1 and VPro2 lead to the formation of 12-membered intramolecularly hydrogen bonded structures very similar to type VI beta-turns with a cis Xaa-VPro amide bond in the major conformers in all the peptides 3-10, resulting in the nucleation of beta-hairpin type structures in these molecules in CDCl(3).

Conformational studies of peptides containing cis-3-hydroxy-D-proline.

Conformational analysis of peptides containing cis-3-hydroxy-d-proline (d-cis-3-Hyp) by NMR studies revealed that the 3-hydroxyl group in this amino acid plays a significant role in the overall three-dimensional structures of the peptides. When the d-cis-3-Hyp had its 3-hydroxyl group protected as the benzyl (Bn) ether, the peptide displayed a beta-hairpin structure in both CDCl(3) and DMSO-d(6). Even after the removal of the Bn group, the resulting deprotected compound retained the same structure as in the protected version in CDCl(3). However, in polar solvent DMSO-d(6), the C-terminal strand of the hydroxyl-deprotected peptide flipped to the side of the hydroxyl group, breaking the hairpin to form a pseudo beta-turn-like nine-membered ring structure involving an intramolecular hydrogen bond between LeuNH --> HypC3-OH.

Studies on novel peptidomimetics having bi-directional dispositions of hydroxylated D-Pro-Gly motifs anchored on a C(2)-symmetric iminosugar-based foundation.

A rigid pyrrolidine based scaffold comprising of 2,5-dideoxy-2,5-imino-D-idaric acid (1) is developed. Attachment of peptide strands to the carboxylic groups at both ends of this novel template led to the peptidomimetics 2 and 3. Conformational analysis by NMR studies revealed that compounds 2b, 3b and 2c, 3c take interesting turn structures (C(2) symmetric for 2c and 3c) in DMSO-d(6) consisting of identical intramolecular hydrogen bonds at two ends between LeuNH --> sugar-OH as depicted in structure A, whereas 2a and 3a display structures with regular beta-turns with hydrogen bonds between LeuNH --> Boc-C=O in one-half of their molecular frameworks (structure B), characteristic of the turn structures commonly observed in "D-Pro-Gly"-containing peptides. These results suggest that a cis hydroxyl group at the 3-position of the proline residue favors a pseudo beta-turn-like nine-membered ring structure in hydroxyproline-containing peptides involving an intramolecular hydrogen bond between the hydroxyl and the i + 2 backbone amide.

Synthesis of a novel cis-proline-derived cyclic type VI beta-turn mimic via ring-closing metathesis.

A cis-proline derived cyclic mimic of a type VI beta-turn is synthesized via a ring-closing metathesis reaction. The solution NMR conformational study indicates that the major conformer of the cyclic peptide adopts a type VIa beta-turn in CDCl(3) and a type VIb beta-turn in DMSO-d(6).

Cyclic homooligomers of furanoid sugar amino acids.

Cyclic homooligomers of mannose-derived furanoid sugar amino acid 1 [H-Maa(Bn(2))-OH] were synthesized by using BOP reagent in the presence of DIPEA under dilute conditions that converted the sugar amino acid monomer directly into its cyclic homooligomers 3a and 4a. The glucose-based sugar amino acid 2 [H-Gaa(Bn(2))-OH] under the same reaction conditions gave a bicyclic lactam 5a as the major product. Cyclic homooligomers of 2 were prepared by cyclizing their linear precursors 6 and 7 leading to the formation of cyclic peptides 8a and 9a, respectively. Conformational analysis by NMR and constrained MD studies revealed that all the cyclic products, 3, 4, 8, and 9, had symmetrical structures. The deprotected cyclic trimer of Maa 3b displayed a conformation in which all the C=O and the N-H bonds of the molecule point in opposite directions. In the deprotected cyclic tetramer of Maa 4b, the COs and NHs were in the plane of the ring with the former pointing to outside and the latter inside the ring. The structure of the cyclic Gaa dimer 8b displayed an unusual six-membered intramolecular hydrogen bond between NH(i)() --> C3-O(i)()(-)(1) and a syn orientation between the C2-H and CO. In this molecule, the C2-hydrogens and the COs can be seen on one side of the ring while the NHs point to the other side. Addition of the bicyclic lactam 5b resulted in the influx of Na(+) ions across the lipid bilayer leading to the dissipation of valinomycin-mediated K(+) diffusion potential.

Robust mixed 10/12 helices promoted by "alternating chirality" in a new family of C-linked carbo-beta-peptides.

The C-linked carbo-beta-peptides, oligomers of a new class of C-linked carbo-beta3-amino acids, have been shown to generate mixed 12/10 and 10/12 helices. The design involves use of "alternating chirality" of the epimeric (at the amine center) monomers to control the stability of these helices. The observation of stable 12/10 helix in a tripeptide and 10/12 helix in a tetrapeptide is unprecedented.

Synthesis and conformational studies of novel cyclic peptides constrained into a 3 10 helical structure by a heterochiral D-pro-L-pro dipeptide template.

An acyclic tripeptide based on a heterochiral D-pro-L-pro template shows a propensity to exist as a 3(10) helical conformation and can be cyclized, via ring-closing metathesis, to the corresponding cyclic tetrapeptides without disrupting the helical conformations in CDCl(3) as well as in DMSO-d(6) solutions. The detailed conformational studies were carried out by using NMR spectroscopy, X-ray crystallography, molecular dynamic simulations, and circular dichroism spectroscopy.