Aspergillus oryzae as a Cell Factory: Research and Applications in Industrial Production.

Aspergillus oryzae, a biosafe strain widely utilized in bioproduction and fermentation technology, exhibits a robust hydrolytic enzyme secretion system. Therefore, it is frequently employed as a cell factory for industrial enzyme production. Moreover, A. oryzae has the ability to synthesize various secondary metabolites, such as kojic acid and L-malic acid. Nevertheless, the complex secretion system and protein expression regulation mechanism of A. oryzae pose challenges for expressing numerous heterologous products. By leveraging synthetic biology and novel genetic engineering techniques, A. oryzae has emerged as an ideal candidate for constructing cell factories. In this review, we provide an overview of the latest advancements in the application of A. oryzae-based cell factories in industrial production. These studies suggest that metabolic engineering and optimization of protein expression regulation are key elements in realizing the widespread industrial application of A. oryzae cell factories. It is anticipated that this review will pave the way for more effective approaches and research avenues in the future implementation of A. oryzae cell factories in industrial production.

Production, purification, properties and current perspectives for modification and application of microbial lipases.

With the industrialization and development of modern science, the application of enzymes as green and environmentally friendly biocatalysts in industry has been increased widely. Among them, lipase (EC. 3.1.1.3) is a very prominent biocatalyst, which has the ability to catalyze the hydrolysis and synthesis of ester compounds. Many lipases have been isolated from various sources, such as animals, plants and microorganisms, among which microbial lipase is the enzyme with the most diverse enzymatic properties and great industrial application potential. It therefore has promising applications in many industries, such as food and beverages, waste treatment, biofuels, leather, textiles, detergent formulations, ester synthesis, pharmaceuticals and medicine. Although many microbial lipases have been isolated and characterized, only some of them have been commercially exploited. In order to cope with the growing industrial demands and overcome these shortcomings to replace traditional chemical catalysts, the preparation of new lipases with thermal/acid-base stability, regioselectivity, organic solvent tolerance, high activity and yield, and reusability through excavation and modification has become a hot research topic.

Geographic Pattern of Variations in Chemical Composition and Nutritional Value of Cinnamomum camphora Seed Kernels from China.

Cinnamomum camphora (camphor tree) is an important non-conventional edible plant species found in East Asia. Here, a detailed characterization for the chemical composition and nutritional value of C. camphora seed kernels (CCSKs) collected from different regions in China is provided. The results showed that there were significant differences among the CCSK samples in weights (1000 fruits, 1000 seeds and 1000 kernels), proximate composition, minerals, phenolics, flavonoids and amino acid contents. The highest contents of oil (62.08%) and protein (22.17%) were found in the CCSK samples collected from Chongqing and Shanghai, respectively. The highest content of mineral in the CCSK samples was K (4345.05-7186.89 mg/kg), followed by P (2735.86-5385.36 mg/kg), Ca (1412.27-3327.37 mg/kg) and Mg (2028.65-3147.32 mg/kg). The CCSK sample collected from Guizhou had the highest levels of total phenolic and flavonoid contents (TPC and TFC), while that from Chongqing had the lowest levels. In addition, the most abundant fatty acid in the CCSK samples was capric acid (57.37-60.18%), followed by lauric acid (35.23-38.29%). Similarities in the fatty acid composition among the CCSK samples were found. The CCSK sample collected from Guizhou had the highest percentage (36.20%) of essential amino acids to total amino acids, and Chongqing had the lowest value (28.84%). These results indicated that CCSK may be developed as an excellent source of plant-based medium-chain oil, protein, dietary fiber, minerals, phytochemicals and essential amino acids.

Biocontrol of Salmonella Typhimurium in milk, lettuce, raw pork meat and ready-to-eat steamed-chicken breast by using a novel bacteriophage with broad host range.

Salmonella spp., one of the most frequently reported bacteria, causes foodborne illness and economic losses. Due to the threat of increasing antibiotic resistant foodborne pathogens, application of bacteriophages as novel antibacterial agents in food matrices has become an emerging strategy. In this study, a novel Salmonella phage PS3-1 with high lytic activity against Salmonella Typhimurium was identified from previously isolated phages. PS3-1 belonged to the class Caudoviricetes with a broad host range, and had relatively short latent period (15 min), large burst size (92 PFU/cell), high pH stability (pH 3.0-11.0) and thermal tolerance (4-60 °C). Genome sequencing analysis showed that PS3-1 genome consisted of 107,110 bp DNA, without antibiotic resistance and virulence related genes. The results of growth curve and time-kill assay showed that PS3-1 not only inhibited the growth of S. Typhimurium, but also effectively decreased the viable cell counts (0.30-4.72 log) after 24-h incubation at 7, 25 and 37 °C (P < 0.05). Moreover, >1.28 log of established biofilm cells were effectively removed after 24-h treatment with PS3-1. Besides, PS3-1 significantly reduced the viability of S. Typhimurium in milk, lettuce, raw pork meat and ready-to-eat steamed-chicken breast at different temperatures (P < 0.05). These results demonstrated that PS3-1 may be an excellent antibacterial agent for controlling S. Typhimurium in food industry.

Dextran Conjugation Improves the Structural and Functional Properties of Heat-Treated Protein Isolate from Cinnamomum camphora Seed Kernel.

The Cinnamomum camphora seed kernel (CCSK), with high contents of medium-chain oil (~59%) and protein (~19%), is an excellent source for a plant-based food ingredient. To broaden the application of the protein isolate (PI) from CCSK in the food industry, the Maillard reaction products (MRPs) were prepared by PI and dextran (DX) under mild wet-heating conditions (60 °C, 5 h), and the structural and functional properties of the PI-DX conjugates were investigated. The covalent bond between PI and DX was confirmed by the degree of grafting and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Compared with the heated PI, the PI-DX conjugates had more ordered structure, with the decreased random coil content. The changes in tertiary structure of PI-DX conjugates were reflected by the results of intrinsic fluorescence and surface hydrophobicity. Moreover, PI-DX conjugates showed better solubility, emulsifying properties, thermal stability and antioxidant activities. These results provided a theoretical basis for the development of PI-based MRPs with desirable characteristics.

Effects of medium chain triglycerides on lipid metabolism in high-fat diet induced obese rats.

This study aimed to compare the effects of three different medium chain triglycerides (MCTs) on lipid metabolism in obese rats. A high fat diet was fed to Sprague-Dawley rats to induce obesity, and then caprylic triglyceride (CYT), capric triglyceride (CT) and lauric triglyceride (LT) were synthesized and used to treat the obese rats for 12 weeks. The obesity phenotype and molecular changes related to lipid metabolism were determined. The results showed that all the three MCTs reduced the body weight (BW) and fat coefficient in obese rats, and the levels of plasma and liver lipids were also improved. Among the MCT groups, the LT group showed the lowest BW with the lowest food intake. Furthermore, three MCTs had different effects on the expression levels of lipid metabolism-related proteins (PPARs and SREBPs). The LT group performed the best among the three MCT groups in the protein expression levels. Interestingly, high-dose LT decreased the expression of the low density lipoprotein receptor (LDLR) in the liver, which impaired the transport of the very low density lipoprotein (VLDL)/low density lipoprotein (LDL) to the liver, resulting in high levels of total plasma cholesterol (TC) and LDL-c in the rats. We have for the first time found that different MCTs had different effects on the expression levels of triacylglycerol and cholesterol metabolism-related proteins in obese rats. These findings would help better understand the relationship between the health benefit and the type of MCT.

Medium-chain triglycerides improve lipid metabolism in obese rats through increasing the browning of adipose tissue via sympathetic regulation.

This study aimed to determine the effect and mechanism of medium-chain triglycerides (MCTs) on promoting the browning of adipose tissue. A high-fat diet was fed to the Sprague-Dawley rats to induce obesity, then caprylic triglycerides (CYTs), capric triglycerides (CTs) and lauric triglycerides (LTs) were used to treat the obese rats for 12 weeks. The obesity phenotype and molecular changes related to browning were determined. The results showed that all three MCTs promoted the browning/beiging of the adipose tissues (brown and white adipose tissue), and lipolysis in these tissues was also increased, which ameliorated high-fat diet-induced lipid metabolic disorders in the obese rats. The LT group showed the highest expression levels of lipolysis- and browning/beiging-related proteins, followed by the CT group and CYT group. We speculate that the browning process may be related to sympathetic regulation and all the three MCT administration increased the secretion of neurotrophic factors both in the hypothalamus and adipose tissue. Then, intra-adipose sympathetic arborization increased, resulting in the activation of the sympathetic regulatory system in MCT groups, and the LT group also showed the highest sympathetic nerve fiber density, followed by the CT group and CYT group. We have for the first time found that the effect of medium-chain fatty acids on adipose tissue browning might be related to the types of fatty acids.

Green synthesis of polydopamine functionalized magnetic mesoporous biochar for lipase immobilization and its application in interesterification for novel structured lipids production.

In this study, the polydopamine functionalized magnetic mesoporous biochar (MPCB-DA) was prepared for immobilization of Bacillus licheniformis lipase via covalent immobilization. Under optimized immobilization conditions, the maximum immobilization yield, efficiency and immobilized lipase amount were found to be 45%, 54% and 36.9 mg/g, respectively. The immobilized lipase, MPCB-DA-Lipase showed good thermal stability and alkali resistance. The MPCB-DA-Lipase retained 56% initial activity after 10 reuse cycles, with more than 85% relative activity after 70 days' storage at 4 or 25 °C. The MPCB-DA-Lipase was efficiently applied in the interesterification of Cinnamomum camphora seed kernel oil and perilla seed oil, with maximum interesterification efficiency of 46%. The produced structured lipids belong to the S2U and U2S triacylglycerols, a novel medium-and long-chain triacylglycerol. These results demonstrated that the MPCB-DA-Lipase may be used as an efficient biocatalyst in lipid processing applications of food industries.

Effects of preheat treatment and polyphenol grafting on the structural, emulsifying and rheological properties of protein isolate from Cinnamomum camphora seed kernel.

In this study, protein isolate (PI) and purified polyphenol extract (PPE) were prepared from Cinnamomum camphora seed kernel (CCSK). The effects of preheat treatment (50-90 °C) combined with polyphenol grafting (5 % PPE, w/w) on the structural, emulsifying and rheological properties of PI were investigated. Results demonstrated the preheat treatments at 80 and 90 °C significantly increased the extent of protein aggregation of PI. Fluorescence spectra and thermal behavior analysis revealed that preheat-treated PI exhibited more compact structure and higher thermal stability. Moreover, the emulsifying stability and apparent viscosity of PI were enhanced after preheat treatments at 50, 60 and 70 °C. After modification by PPE, the secondary structural changes of preheat-treated PI were confirmed by FTIR. PPE modification improved the thermal stability and antioxidant activities of preheat-treated PI. These results provide a novel way to combine the advantages of preheat treatment and polyphenol grafting in developing a novel protein ingredient.

Immobilization of lipase on ß-cyclodextrin grafted and aminopropyl-functionalized chitosan/Fe3O4 magnetic nanocomposites: An innovative approach to fruity flavor esters esterification.

The lipase from Bacillus licheniformis NCU CS-5 was immobilized onto ß-cyclodextrin (CD) grafted and aminopropyl-functionalized chitosan-coated Fe3O4 magnetic nanocomposites (Fe3O4-CTS-APTES-GA-ß-CD). Fourier transform infrared spectroscopy, thermogravimetry analysis, X-ray diffraction, scanning electron microscopy and transmission electron microscopy showed that not only the functionalized magnetic nanoparticles were synthesized but also the immobilized lipase was successfully produced. The immobilized lipase exhibited higher optimal pH value (10.5) and temperature (60℃) than the free lipase. The pH and thermal stabilities of the immobilized lipase were improved significantly compared to the free lipase. The immobilized lipase remained more than 80% of the relative activity at temperature of 60 ℃ and pH 12.0. The immobilized lipase also remained over 80% of its relative activity after 28 days of storage and 15 cycles of application. The application of the immobilized lipase in esterification of isoamyl acetate and pentyl valerate showed that maximum esterification efficiency was achieved in n-hexane having 68.0% and 89.2% respectively. Therefore, these results indicated that the Fe3O4-CTS-APTES-GA-ß-CD nanoparticles are novel carriers for immobilizing enzyme, and the immobilized lipase can be used as an innovative green approach to the synthesis of fruity flavor esters in food industry.

Expression and characterization of a novel lipase from Bacillus licheniformis NCU CS-5 for application in enhancing fatty acids flavor release for low-fat cheeses.

A novel lipase from Bacillus licheniformis NCU CS-5 was expressed in different Escherichia coli cells. The recombinant enzyme achieved a high activity (161.74 U/mL) with protein concentration of 0.27 mg/mL under optimal conditions at the large-scale expression of 12 h. The recombinant lipase showed optimal activity at 40 ℃ and pH 10.0, and maintained more than 80% relative activity after 96 h of incubation at pH 9.0-10.0. This typical alkaline lipase was activated under medium temperature conditions (30 and 45 ℃ for 96 h). The lipase exhibited a degree of adaptability in various organic solvents and metal ions, and showed high specificity towards triglycerides with short and medium chain fatty acids. Among different substrates, the lipase showed the strongest binding affinity towards pNPP (Km = 0.674 mM, Vmax = 950.196 µM/min). In the experiments of its application in enhancing fatty acids flavor release for low-fat cheeses, the lipase was found to hydrolyze cheeses and mainly increase the contents of butyric acid, hexanoic acid, caprylic acid and decanoic acid. The results from NMR and GC provided the possibility of enhancing fatty acids flavor released from low-fat cheeses by the lipolysis method.

Assessment of the effect of ethanol extracts from Cinnamomum camphora seed kernel on intestinal inflammation using simulated gastrointestinal digestion and a Caco-2/RAW264.7 co-culture system.

Cinnamomum camphora seeds have multiple bioactivities. There were few studies on the effect of C. camphora seeds on intestinal inflammation in vitro and in vivo. The study aimed to investigate the effects of ethanol extracts from C. camphora seed kernel on intestinal inflammation using simulated gastrointestinal digestion and a Caco-2/RAW264.7 co-culture system. Results showed that the digested ethanol extracts (dEE) were rich in polyphenols, and a total of 17 compounds were tentatively identified using UPLC-LTQ-Orbitrap-MS/MS. dEE increased cell viability, while decreasing the production of reactive oxygen species, and the secretion and gene expression of inflammatory markers (NO, PGE2, TNF-α, IL-1ß and IL-6). dEE also down-regulated NF-κB/MAPK pathway activities by suppressing the phosphorylation of relevant signaling molecules (p65, IκBα, ERK and p38), as well as the expression of TLR4 receptor protein. Furthermore, dEE may improve intestinal barrier function by increasing the TEER value, and the expression of tight junction proteins (ZO-1, claudin-1 and occludin). The results suggest the ethanol extracts from C. camphora seed kernel may have strong anti-inflammatory activities, and a potential application in the prevention or treatment of intestinal inflammation and enhancement of intestinal barrier function in organisms.

High Dietary Intervention of Lauric Triglyceride Might be Harmful to Its Improvement of Cholesterol Metabolism in Obese Rats.

Hypercholesterolemia is often considered to be a major risk factor for atherosclerosis, and medium-chain fatty acids have been found to reduce the total cholesterol (TC) level and maintain low-density lipoprotein cholesterol (LDL-c) stability. However, we unexpectedly found that the levels of TC and LDL-c were increased in obese rats treated with high-dose lauric triglycerides (LT). The study aimed to investigate the effect and mechanism of LT on cholesterol metabolism in obese rats. Our results showed that LT intervention could reduce cholesterol biosynthesis by downregulating the expression of HMG-CoA reductase in obese rats. LT increased the expression levels of PPARγ1, LXRα, ABCA1, and ABCG8 in the liver. These results indicated that LT could improve the lipid transfer and bile acid efflux. However, LT significantly increased the expression of PCSK 9, resulting in accelerated degradation of LDLR, thus reducing the transport of very LDL (VLDL) and LDL to the liver. Together with the increased expression of NPC1L1 protein, LT impaired the uptake of VLDL/LDL by the liver and increased the reabsorption of sterols, leading to an increase in the levels of TC and LDL-c in obese rats.

Characterization of a novel lipase from Bacillus licheniformis NCU CS-5 for applications in detergent industry and biodegradation of 2,4-D butyl ester.

Enzymatic degradation has become the most promising approach to degrading organic ester compounds. In this study, Bacillus licheniformis NCU CS-5 was isolated from the spoilage of Cinnamomum camphora seed kernel, and its extracellular lipase was purified, with a specific activity of 192.98 U/mg. The lipase was found to be a trimeric protein as it showed a single band of 27 kDa in SDS-PAGE and 81 kDa in Native-PAGE. It was active in a wide range of temperatures (5-55 °C) and pH values (6.0-9.0), and the optimal temperature and pH value were 40 °C and 8.0, respectively. The enzyme was active in the presence of various organic solvents, metal ions, inhibitors and surfactants. Both crude and purified lipase retained more than 80% activity after 5 h in the presence of commercial detergents, suggesting its great application potential in detergent industry. The highest activity was found to be towards medium- and long-chain fatty acids (C6-C18). Peptide mass spectrometric analysis of the purified lipase showed similarity to the lipase family of B. licheniformis. Furthermore, it degraded more than 90% 2,4-D butyl ester to its hydrolysate 2,4-D within 24 h, indicating that the novel lipase may be applied to degrade organic ester pesticides.

Lauric Triglyceride Ameliorates High-Fat-Diet-Induced Obesity in Rats by Reducing Lipogenesis and Increasing Lipolysis and ß-Oxidation.

Medium-chain triglycerides (MCTs) are found in limited foods. In these medium-chain oil resources, the abundance of lauric acid (LA) is the highest among medium-chain fatty acids (MCFAs), and its effects on lipid metabolism in obese rats have not been well-studied. This study aimed to determine the anti-obesity effects and mechanisms of lauric triglyceride (LT) in Sprague Dawley (SD) rats. LA and glycerin were used to synthesize LT, then LT was used to treat obese rats for 12 weeks. The results showed that LT significantly reduced the body weight, body mass index, and Lee's index in obese rats. The mRNA expression levels of the anorexic neuropeptide POMC in the hypothalamus between the LT group and the other groups were not different, while the gene expression levels of the orexigenic neuropeptides NPY and AGRP decreased significantly in the LT group. Except serum cholesterol, LT improved the serum triglyceride metabolism in the obese rats and reduced adipocyte and hepatic lipid deposition. Moreover, LT inhibited the expression of lipogenesis-related genes and proteins (SREBP-1c, ACC1, and FASN) and increased the expression of lipolysis (ATGL, HSL, and LPL) and ß-oxidation (PPARα, CPT-1a, and PCG-1α) related genes and proteins in the white fat and liver. Furthermore, LT increased the mRNA expression of mitochondrial-biosynthesis-related genes (SIRT1, NRF1, and TFAM) in the liver. The results indicated that LT ameliorates diet-induced obesity in rats.

Production, purification and biochemical characterisation of a novel lipase from a newly identified lipolytic bacterium Staphylococcus caprae NCU S6.

A novel lipase, SCNL, was isolated from Staphylococcus caprae NCU S6 strain in the study. The lipase was purified to homogeneity with a yield of 6.13% and specific activity of 502.76 U/mg, and its molecular weight was determined to be approximately 87 kDa. SCNL maintained above 80% of its initial activity at a wide range of temperatures (20-50 °C) and pH values (6-11), with an optimal temperature at 40 °C and optimal pH at 9.0 with p-nitrophenyl palmitate as a substrate. SCNL exhibited a higher residual activity than the other staphylococcal lipases in the presence of common enzyme inhibitors and commercial detergents. The lipase activity was enhanced by organic solvents (isooctane, glycerol, DMSO and methanol) and metal ions (Na+, Ba2+, Ca2+, and Mn2+). The Km and Vmax values of SCNL were 0.695 mM and 262.66 s-1 mM-1, respectively. The enzyme showed a preference for p-NP stearate, tributyrin and canola oil. These biochemical features of SCNL suggested that it may be an excellent novel lipase candidate for industrial and biotechnological applications.

Ethanol extracts from Cinnamomum camphora seed kernel: Potential bioactivities as affected by alkaline hydrolysis and simulated gastrointestinal digestion.

The aim of the study was to evaluate the changes of potential bioactivities of ethanol extracts (EE) from Cinnamomum camphora seed kernel (CCSK) after alkaline hydrolysis and simulated gastrointestinal digestion. A total of 13 compounds in EE, mainly phenolics and saponins were tentatively identified using HPLC-ESI-QTOF-MS2 analysis. The total phenolic and total flavonoid contents in EE decreased by 30.6%, 1%, 33% and 11.8% after hydrolysis and digestion, respectively. The total saponins content decreased by 17% after hydrolysis while increased by 48% after digestion. The total condensed tannin contents increased by 70.3% and 17.2% after hydrolysis and digestion, respectively. The 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid (ABTS), cupric ion reducing activity (CUPRAC), ferric reducing antioxidant power (FRAP) and metal chelating activity (MCA) were used to determine antioxidant activity. Overall, the changes of antioxidant activities by hydrolysis and digestion were consistent with the changes of their total phenolic and flavonoid contents. The α-amylase and α-glucosidase inhibitory activities in EE increased by 817% and 33.3% after digestion while decreased by 12.3% and 19% after hydrolysis, respectively. Although the inhibitory activities of cholinesterase, tyrosinase and xanthine oxidase were decreased by hydrolysis and digestion, most of these activities were retained. These results showed that CCSK ethanol extracts had strong bioactivities and were reasonably stable to alkali and digestive enzymes.

Effects of medium- and long-chain fatty acids on acetaminophen- or rifampicin-induced hepatocellular injury.

Drug-induced liver injury (DILI) is one of the common adverse effects of drug therapy, which is closely associated with oxidative stress, apoptosis, and inflammation response. Medium-chain fatty acids (MCFA) were reported to relieve inflammation and attenuate oxidative stress. However, little has been known about the hepatoprotective effects of MCFA in DILI. In the present study, acetaminophen (AP) and rifampicin (RFP) were used to establish DILI models in LO2 cells, and the cytoprotective effects of MCFA on hepatocellular injury were investigated. Results showed that the optimal condition for the DILI model was treatment with 10 mM AP or 600 µM RFP for 24 hr. LCFA treatment markedly reduced the cell viability and increased the activities of alanine aminotransferase, aspartate aminotransferase, and lactate dehydrogenase. Meanwhile, LCFA treatment aggravated cell apoptosis, mitochondrial dysfunction, and oxidative stress. The mRNA and protein expression levels of inflammatory cytokines (IL-1ß and TNF-α) were significantly elevated by LCFA. In contrast, MCFA treatment did not significantly affect cell viability, apoptosis, oxidative, stress and inflammation, and it did not produce the detrimental effects on DILI models. Therefore, we proposed that MCFA may be more safe and suitable than LCFA as nutrition support or the selection of daily dietary oil and fat for the patients with DILI.

In Vitro Antibacterial Activity and Mechanism of Monocaprylin against Escherichia coli and Staphylococcus aureus.

In the present study, the antibacterial activity of monocaprylin in comparison with sodium benzoate and potassium sorbate against Staphylococcus aureus and Escherichia coli was assessed by measuring MIC, MBC, effect of pH on MIC, and incubation temperature on bactericidal efficacy. Results showed that monocaprylin exhibited an excellent antibacterial activity against both strains, with the lowest MIC and MBC of 1.28 mg/mL. A MIC of monocaprylin remained unchanged despite the pH values of culture medium, ranging from 5 to 9, unlike that of potassium sorbate or sodium benzoate. Furthermore, monocaprylin at MBC effectively reduced the population of E. coli and S. aureus by >5.5 log CFU/mL at 25°C within 6 h and decreased E. coli by approximately 5.0 log CFU/mL and S. aureus by 2.9 log CFU/mL at 12 h. The underlying mechanism of monocaprylin was then investigated by measuring ß-galactosidase activity, membrane potential, release of cellular contents, scanning electron microscopy, and transmission electron microscopy observations. Results indicated that monocaprylin killed E. coli by the rapid change in permeability and integrity of cell membrane, leading to decline of membrane potential, leakage of nucleic acids and proteins, and ultimately cell membrane disintegration and lysis. On the other hand, monocaprylin might exert its antibacterial activity against S. aureus mainly by diffusing across the cell wall, collapsing the cell membrane, and disturbing the order of intracellular contents. These findings indicated that monocaprylin had better antibacterial ability compared with traditional synthetic preservatives and might be a potential antibacterial additive independent of pH.

Mutagenesis and characterization of a Bacillus amyloliquefaciens strain for Cinnamomum camphora seed kernel oil extraction by aqueous enzymatic method.

The purpose of the present study was to increase the proteinase activity of the strain NCU116 by combining ultraviolet irradiation and N-methyl-N'-nitro-N-nitroso guanidine treatment, in order to enhance the efficiency of Cinnamomum camphora seed kernel oil (CCSKO) extraction by aqueous enzymatic method (AEM). The mutated strain, designated as NCU116-1, was screened out by the ratio of hydrolytic zone diameter to colony diameter on skim milk plate. The proteinase activity (9116.1 U/ml) of NCU116-1 was increased by 31.9% compared with the parental strain. The extracellular enzymes produced by NCU116-1 included proteinase, pectase, glucoamylase, cellulase and amylase. The proteinase had the maximum activity at 50 °C. Its optimum temperature and pH value were approximately 45 °C and 8.0 respectively. Mn2+ was an activator of neutral proteinase. The glucoamylase had the maximum activity at 35 °C, and was activated by Cu2+, Fe3+ and Mn2+. Its optimum temperatures and pH value were 35 °C and 8.0 respectively. The pectinase had the maximum activity at 40 °C, and was activated by Ca2+ and Mn2+. Its optimum temperatures and pH value were 35-40 °C and 6.0 respectively. The optimum conditions of CCSKO extraction by AEM were also investigated. The results suggested that the best amount of enzyme solution and enzymolysis time were 20% (v/v) and 4 h, respectively. The oil extraction rate was 95.2% under these conditions. Thus, a suitable mutated strain was selected for CCSKO extraction by AEM and the optimum extraction conditions were determined.