Safety Evaluation and Whole Genome Sequencing of Aspergillus japonicas PJ01 Reveal Its Potential to Degrade Citrus Segments in Juice Processing

Aspergillus japonicas PJ01 (A. japonicas PJ01) is a strain isolated from the rotten branches. In previ-ous studies, it was shown that it can produce complex enzymes to degrade polysaccharide com-ponents. In this study, we evaluated the safety of its crude enzyme solution. Acute oral toxicity, subchronic toxicity, micronucleus and sperm malformation tests all validated the high biologi-cal safety for the crude enzymes. Secondly, we carried out the citrus segment degradation ex-periment of crude enzyme solution. Compared with the control group, the crude enzyme solu-tion of A. japonicas PJ01 can completely degrade the segments in 50 min, which provides the basis for enzymatic peeling during juice processing. The whole genome sequencing showed that the genome of A. japonicus PJ01 has a GC content of 51.37% with a size of 36204647 bp, and encoded 10070 genes. GO, COG, KEGG and CAZy databases were used in gene annotation analyses. Pathway enrichment showed many genes related to carbohydrate metabolism, rich in genes re-lated to pectinase, xylanase and carboxylcellulase. Therefore, the complex enzyme produced by A. japonicus PJ01 can be used in gizzard juice processing to achieve efficient enzymatic decapsu-lation.

Enzyme-Assisted Method for Phycobiliproteins Extraction from Porphyra and Evaluation of Their Bioactivity

Due to the poor protein availability of algae in their unprocessed form, development of extraction methods for phycobiliproteins is of great significance. This study aimed to extract phycoerythrin (PE) and phycocyanin (PC) from Porphyra via bacterial enzymatic hydrolysis and to evaluate their bioactivity. To induce enzyme production, Porphyra powder was added into the culture medium of two marine bacterial strains. The pH and enzyme activity of the cultured supernatant, namely crude enzyme solution, were significantly raised. For PE and PC extraction, Porphyra were incubated within crude enzyme solution with homogenization and ultrasonication followed by ultrafiltration process. After distinguishing by fast performance liquid chromatography (FPLC), three major fractions were observed and identified as R-PE, R-PC and small molecular PE by high-performance liquid chromatography (HPLC) and polyacrylamide gel electrophoresis (PAGE) analysis. With respect to bioactivity, these three fractions exhibited free radical scavenging and antioxidant activities in a various degree. In addition, the angiotensin-converting-enzyme (ACE) inhibitory activity of both R-PE and R-PC fractions was observed in a concentration-dependent manner. Taken together, the employed process of bacterial enzymatic hydrolysis is suggested to be a feasible method to obtain PE and PC from Porphyra without limiting their bioactivity.

Preparation and immobilization Glucoamylase and Pectinase by CLEA method

CLEA method (cross-linking enzyme aggregates) combines enzyme preparation and immobilization from solution culture into the same step. In this study, we applied CLEA method to immobilize two enzymes, glucoamylase and pectinase, from crude enzyme solution obtained from semi-solid culture of Aspergillus niger. The results showed that: In the same immobilized conditions (glucoamylase: 7% glutaraldehyde, 5°C, 2 hours; pectinase: 10% glutaraldehyde, 5°C, 2 hours), the immobilized enzyme from crude enzyme solution, has the abilities to be reused and activation stability under the influences of pH and temperature higher than immobilized commercial enzyme respectively.

Immobilized Recombinant Chitinase and its Inhibition Effect on Botrytis Cinerea

To isolate recombinant chitinase quickly and boost its anti-fungi activities in vitro, functional magnetic nanometer carrier was used to immobilize recombinant chitinase from the crude enzyme solution and immobilized recombinant chitinase was applied to test whether it would inhibit the growth of gray mold from fruits. In this study, the carboxyl magnetic carrier was produced by solvent thermal reduction method and characterized by scanning electron microscope (SEM) and fourier transform infrared spectrometer (FTIR). Then, the carboxyl magnetic carrier activated by EDC/NHS was applied to immobilize recombinant chitinase and the immobilization efficiency was investigated by quantitative analysis. To obtain the highest immobilization efficiency, reaction conditions were optimized through combining different pH, temperature and reaction period. The results show that the surface of magnetic carrier was successfully carboxyl and the average diameter was 200nm. The immobilization efdiciency could reach the peak 64.43% after 7h reaction at the condition of pH 6 and 25°C. It also shows that immobilized recombinant chitinase can significantly inhibit the growth of gray mold isolated from table grape compared with the enzyme without immobilization with magnetic nanometer carrier.

Purification and Characterization of Ginsenoside-Hydrolyzing β-Glucosidase from Wheat Bran

The ginsenoside-hydrolyzing β-glucosidase that can converse the major ginsenosides into the minor ginsenosides was isolated from wheat bran, and the enzyme was purified and characterized. The crude enzyme solution extracted from wheat bran could hydrolyse the protopanaxadiol-type ginsenosides such as Rb1, Rc, Rd and Rg3, but could not hydrolyse the protopanaxatriol-type ginsenosides such as Re and Rg2. The enzyme fractionated on the DEAE-Cellulose DE-52 column was purified to one spot in SDS polyacrylamide gel electrophoresis, and the molecular weight of enzyme in the fraction 34, 47, and 61 was approximately 62 kDa, 62 kDa, and 68 kDa, respectively.

Production and properties of Xylanase from thermophilic Bacillus sp.

An aerobic, thermophilic, xylanolytic bacterium was isolated from local soil. The results of 16S rRNA sequence comparisons indicated that the isolate was closely related to Bacillus caldoxylolyticus and Bacillus sp strain AK1. These organisms exhibited 94% levels of ribossomal DNA sequence homology. Studies on the xylanase characterisation from liquid cultures grown on beechwood xylan revealed that the enzyme retained 100% of activity for 2 hours at temperatures ranging from 30 to 50º C, while at 60, 70 and 100º C, 10%, 11% and 29% of the original activities were lost, respectively. The optimum pH of the enzyme was found to be between 6.5 and 7.0. After incubation of crude enzyme solution for 24 hours at 25º C and at pH 5.5 to 8.0, a decrease of about 12% of its original activity was observed.