Investigation of Structural Features of Two Related Lipases and the Impact on Fatty Acid Specificity in Vegetable Fats.

One of the indispensable applications of lipases in modification of oils and fats is the possibility to tailor the fatty acid content of triacylglycerols (TAGs), to meet specific requirements from various applications in food, nutrition, and cosmetic industries. Oleic acid (C18:1) and stearic acid (C18:0) are two common long fatty acids in the side chain of triglycerides in plant fats and oils that have similar chemical composition and structures, except for an unsaturated bond between C9 and C10 in oleic acid. Two lipases from Rhizomucor miehei (RML) and Rhizopus oryzae (ROL), show activity in reactions involving oleate and stearate, and share high sequence and structural identity. In this research, the preference for one of these two similar fatty acid side chains was investigated for the two lipases and was related to the respective enzyme structure. From transesterification reactions with 1:1 (molar ratio) mixed ethyl stearate (ES) and ethyl oleate (EO), both RML and ROL showed a higher activity towards EO than ES, but RML showed around 10% higher preference for ES compared with ROL. In silico results showed that stearate has a less stable interaction with the substrate binding crevice in both RML and ROL and higher tendency to freely move out of the substrate binding region, compared with oleate whose structure is more rigid due to the existence of the double bond. However, Trp88 from RML which is an Ala at the identical position in ROL shows a significant stabilization effect in the substrate interaction in RML, especially with stearate as a ligand.

Co-Immobilization of Rhizopus oryzae and Candida rugosa Lipases onto mMWCNTs@4-arm-PEG-NH2-A Novel Magnetic Nanotube-Polyethylene Glycol Amine Composite-And Its Applications for Biodiesel Production.

This article describes the successful synthesis of a novel nanocomposite of superparamagnetic multi-walled nanotubes with a four-arm polyethylene glycol amine polymer (mMWCNTs@4-arm-PEG-NH2). This composite was then employed as a support for the covalent co-immobilization of Rhizopus oryzae and Candida rugosa lipases under appropriate conditions. The co-immobilized lipases (CIL-mMWCNTs@4-arm-PEG-NH2) exhibited maximum specific activity of 99.626U/mg protein, which was 34.5-fold superior to that of free ROL, and its thermal stability was greatly improved. Most significantly, CIL-mMWCNTs@4-arm-PEG-NH2 was used to prepare biodiesel from waste cooking oil under ultrasound conditions, and within 120 min, the biodiesel conversion rate reached 97.64%. This was due to the synergy effect between ROL and CRL and the ultrasound-assisted enzymatic process, resulting in an increased biodiesel yield in a short reaction time. Moreover, after ten reuse cycles, the co-immobilized lipases still retained a biodiesel yield of over 78.55%, exhibiting excellent operational stability that is attractive for practical applications. Consequently, the combined use of a novel designed carrier, the co-immobilized lipases with synergy effect, and the ultrasound-assisted enzymatic reaction exhibited potential prospects for future applications in biodiesel production and various industrial applications.

Development of a novel multi-strain wheat Qu with high enzyme activities for Huangjiu fermentation.

BACKGROUND: Wheat Qu has long been used as a fermentation starter to produce Huangjiu. Wheat Qu quality depends on its microbial community structure and the hydrolytic enzymes generated by the micro-organisms. RESULTS: Strain YF1 and YF2 were successfully screened as they exhibited high acidic protease (231.9 ± 1.4 U g-1 ) and cellulase (7.1 ± 0.6 U g-1 ) activities. Based on a morphological and sequence analysis of the internal transcribed spacer (ITS) gene, YF1 and YF2 were identified as Rhizopus oryzae and Aspergillus niger, respectively. Cooked wheat Qu was produced using mixed fungal starter fermentations with Aspergillus oryzae SU-16, YF1, and YF2. For Qu-making, the optimized conditions for fermentation time, water content, and inoculum size were 47.8 h, 69.4%, and 6.1%, respectively. Under these conditions, compared with single-strain cooked wheat Qu, enzyme activities of amylase, acidic protease, and cellulase increased by 27.4%, 657.1%, and 1276.2%, respectively. Short peptides and free amino acids contents increased by 19.6% and 131.8%, respectively. This wheat Qu was used for Huangjiu brewing, and the alcohol content increased by approximately 14.6% because of the increased starch hydrolysis efficiency mainly attributed to its high enzyme activity. CONCLUSION: Using mixed fungal strains as starter cultures may be an efficient strategy to improve wheat Qu quality, with great potential for application in industrial Huangjiu production. © 2021 Society of Chemical Industry.

Domesticating a food spoilage yeast into an organic acid-tolerant metabolic engineering host: Lactic acid production by engineered Zygosaccharomyces bailii.

Lactic acid represents an important class of commodity chemicals, which can be produced by microbial cell factories. However, due to the toxicity of lactic acid at lower pH, microbial production requires the usage of neutralizing agents to maintain neutral pH. Zygosaccharomyces bailii, a food spoilage yeast, can grow under the presence of organic acids used as food preservatives. This unique trait of the yeast might be useful for producing lactic acid. With the goal of domesticating the organic acid-tolerant yeast as a metabolic engineering host, seven Z. bailii strains were screened in a minimal medium with 10 g/L of acetic, or 60 g/L of lactic acid at pH 3. The Z. bailii NRRL Y7239 strain was selected as the most robust strain to be engineered for lactic acid production. By applying a PAN-ARS-based CRISPR-Cas9 system consisting of a transfer RNA promoter and NAT selection, we demonstrated the targeted deletion of ADE2 and site-specific integration of Rhizopus oryzae ldhA coding for lactate dehydrogenase into the PDC1 locus. The resulting pdc1::ldhA strain produced 35 g/L of lactic acid without ethanol production. This study demonstrates the feasibility of the CRISPR-Cas9 system in Z. bailii, which can be applied for a fundamental study of the species.

Synthesis of propyl benzoate by solvent-free immobilized lipase-catalyzed transesterification: Optimization and kinetic modeling.

The present study aimed to analyze reaction kinetics and mechanism for the synthesis of propyl benzoate in solvent-free conditions. Lipase was immobilized on Hydroxypropyl methylcellulose (HPMC) and polyvinyl alcohol (PVA) polymer blend by entrapment method. Among different lipases immobilized on a support, Candida cylindracea (CCL) showed excellent activity. Systematic studies were done to optimize the reaction conditions. The activation energy was found to be 16.2 kcal/mol for immobilized CCL. Kinetic parameters were calculated, which depicted that propyl benzoate synthesized using immobilized CCL followed the ternary complex model in which propanol inhibits lipase activity at higher concentrations. Recyclability of the catalyst was checked up to four catalytic cycles and 40% retention of activity was observed up to the fourth cycle. Finally, the applicability of developed protocol to synthesize various alkyl benzoates was explored.

Improving the thermostability and acid resistance of Rhizopus oryzae α-amylase by using multiple sequence alignment based site-directed mutagenesis.

Higher thermostability or acid resistance for fungal α-amylase will help to improve the sugar-making process and cut down the production costs. Here, the thermostability or acid resistance of Rhizopus oryzae α-amylase (ROAmy) was significantly enhanced by site-directed evolution based on multiple sequence alignment (MSA) method. For instance, compared with the wild-type ROAmy, the optimum temperature of mutants G136D and A144Y was increased from 50 to 55 °C, whereas for mutants V174R and I276P, the optimum temperature was increased from 50 to 60 °C. The optimum pH of mutants G136D and A144Y shifted from 5.5 to 5.0, whereas for mutants V174R and T253E, the optimum pH changed from 5.5 to 4.5. The results showed that mutant V174R had a 2.52-fold increase in half-life at 55 °C, a 2.55-fold increase in half-life at pH 4.5, and a 1.61-fold increase in catalytic efficiency (kcat /Km ) on soluble starch. The three-dimensional model simulation revealed that changes of hydrophilicity, hydrogen bond, salt bridge, or rigidity observed in mutants might mainly account for the improvement of thermostability and acid resistance. The mutants with improved catalytic properties attained in this work may render an accessible and operable approach for directed evolution of fungal α-amylase aimed at interesting functions.

Bio characterization of purified isoamylase from Rhizopus oryzae.

Extracellular isoamylase produced by Rhizopus oryzae PR7 MTCC 9642 in in Erlenmeyer flasks was purified by ultrafiltration and by two steps of Superose 6 C-10/300GL gel chromatography. The enzyme molecule was found to be a monomer with molecular weight of 68 kDa.The purified isoamylase showed optimum activity at pH 5.5 and temperature 55 °C. The catalytic activity was found to remain stable at a broad range of pH (4-8) and could show remarkable thermo resistance specially in presence of exogenous thiols. The noteworthy enhancement of activity in presence of Mn2+ indicated its role as enzyme cofactor while thermos and chemostability in presence of exogenous thiols indicated the presence of disulfide linkage at active site of the enzyme. Both in vitro study and doking analysis indicated the highest affinity of the isoamylase of R. oryzae PR7 toward glycogen and the enzyme exhibited Km and Vmax values of 0.38 mg/mL and 6.65 mM/min/mL, respectively. Purified debranching amylolytic enzyme from R. oryzae PR7 has potential for the study of glycogen and starch structure and industrial application in combination with other amylolytic enzymes. The rapid, convenient, relatively simple purification process and other functional attributes of the enzyme made it competent to be employed for industrial utilization.

Purification and characterization of an aspartic protease from the Rhizopus oryzae protease extract, Peptidase R

Background Aspartic proteases are a subfamily of endopeptidases that are useful in a variety of applications, especially in the food processing industry. Here we describe a novel aspartic protease that was purified from Peptidase R, a commercial protease preparation derived from Rhizopus oryzae. Results An aspartic protease sourced from Peptidase R was purified to homogeneity by anion exchange chromatography followed by polishing with a hydrophobic interaction chromatography column, resulting in a 3.4-fold increase in specific activity (57.5 × 10³ U/mg) and 58.8% recovery. The estimated molecular weight of the purified enzyme was 39 kDa. The N-terminal sequence of the purified protein exhibited 63-75% identity to rhizopuspepsins from various Rhizopus species. The enzyme exhibited maximal activity at 75°C in glycine-HCl buffer, pH 3.4 with casein as the substrate. The protease was stable at 35°C for 60 min and had an observed half-life of approximately 30 min at 45°C. Enzyme activity was not significantly inhibited by chelation with ethylenediamine tetraacetic acid (EDTA), and the addition of metal ions to EDTA-treated protease did not significantly change enzyme activity, indicating that proteolysis is not metal ion-dependent. The purified enzyme was completely inactivated by the aspartic protease inhibitor Pepstatin A. Conclusion Based on the observed enzyme activity, inhibition profile with Pepstatin A, and sequence similarity to other rhizopuspepsins, we have classified this enzyme as an aspartic protease.