Zhongyang Ding

A Newly Isolated Organic Solvent Tolerant Staphylococcus saprophyticus M36 Produced Organic Solvent-Stable Lipase

Thirty-eight high lipase activity strains were isolated from soil, seawater, and Brassica napus. Among them, a novel organic solvent tolerant bacterium (strain M36) was isolated from the seawater in Jiangsu, China. Isolate M36 was able to grow at high concentration of benzene or toluene up to 40% (vol/vol), and later identified as Staphylococcus saprophyticus by biochemical test and 16s ribosomal DNA sequence. No work on Staphylococcus producing lipase with organic solvent tolerance has been reported so far. The lipase of strain M36 whose activity in liquid medium was 42 U mL−1 at 24-h incubation time was stable in the presence of 25% (vol/vol) p-xylene, benzene, toluene, and hexane.

Production of milk-clotting enzyme by Bacillus subtilis B1 from wheat bran

Three strains, Bacillus subtilis B1, B. subtilis B18 and Bacillus thuringiensis B12, were screened from wheat bran to produce milk-clotting enzyme. Among them, B. subtilis B1 exhibited considerable milkclotting activity with low proteolytic activity. After response surface methodology optimization, milkclotting activity was improved from 782 SU ml -1 to 1129.05 ± 74.55 SU mL -1 with a small inoculum size (0.130%, v/v). The optimized medium contained glucose (16.2 gL -1 ), wheat bran (30 gL -1 ), NaCl (5 gL -1 ), MgSO 4 .7H 2 O (5 gL -1 ), KH 2 PO 4 (2 gL -1 ) and CaCO 3 (3 gL -1 ). The milk-clotting enzyme from B. subtilis B1 has the optimum pH 5.5 and CaCl 2 concentration 50 mmol L -1 . It is completely inactivated after 5 min at 70°C. The result showed that B. subtilis B1 was a promising strain for industrial milk-clotting enzyme production. Key words : Milk-clotting enzyme, Bacillus subtilis, submerged fermentation, wheat bran.

Effects of culture conditions on monosaccharide composition of Ganoderma lucidum exopolysaccharide and on activities of related enzymes.

We investigated the relationship between monosaccharide composition of Ganoderma lucidum exopolysaccharide (EPS) and activities of EPS synthesis enzymes under various culture temperatures and initial pH values. The mole percentages of three major EPS monosaccharides, glucose, galactose and mannose, varied depending on culture conditions and the resulting EPS displayed differing anti-tumor activities. In nine tested enzymes, higher enzyme activities were correlated with higher temperature and lower initial pH. Altered mole percentages of galactose and mannose under various culture conditions were associated with activities of α-phosphoglucomutase (PGM) and phosphoglucose isomerase (PGI), respectively, and that of mannose was also associated with phosphomannose isomerase (PMI) activity only under various pH. Our findings suggest that mole percentages of G. lucidum EPS monosaccharides can be manipulated by changes of culture conditions that affect enzyme activities, and that novel fermentation strategies based on this approach may enhance production and biological activity of EPS.

Polysaccharides production by submerged fermentation of Coprinus comatus and their inhibitory effects on non-enzymatic glycosylation

Bioactive polysaccharides produced by fungi, especially by medicinal mushrooms, have attracted great interest. Polysaccharides from Coprinus comatus have been reported to possess hypoglycemic activity. In the present study, the effects of carbon source, initial glucose concentration and initial peptone level on cell growth and polysaccharide production were examined in the submerged fermentation system of C. comatus. Maltose was found to be disadvantageous for cell growth, but favorable for extracellular polysaccharide (EPS) production. Glucose was beneficial for both cell growth and production of intracellular polysaccharide (IPS), which was markedly improved when a high initial glucose concentration was used. The highest maximal EPS production level (0.89 g/l) was obtained with an initial glucose concentration of 40 g/l. Cell growth was positively related to increases in initial peptone concentration and polysaccharide production was maximized at 10 g/l of peptone. Finally, high inhibitory activity of Coprinus polysaccharides (both EPS and IPS) on non-enzymatic glycosylation (NEG) was demonstrated for the first time. The findings in this study can be applied to improve the performance of C. comatus cultures in the production of bioactive metabolites on a bioreactor scale and provide a foundation for further investigation into medicinally active compounds derived from C. comatus.

High-efficient production of citric acid by Aspergillus niger from high concentration of substrate based on the staged-addition glucoamylase strategy

Citric acid (CA), an important platform-compound, has attracted much attention because of its broad applications and huge market demand. To solve high residual sugar at the fermentation end, we put forwarded strategy of pre-saccharification and then fermentation. Results showed that the residual total sugar decreased by 10.4% and the productivity increased by 4.0% and initially high glucose inhibited cell growth. Furthermore, commercial glucoamylase with high low-pH stability was proposed to staged-add in the fermentation process, which timely compensated enzyme loss, ensuring the glucose supply rate. The fermentation productivity was evidently enhanced by 13.3% with residual total sugar decreasing by 31.3%, simplifying the subsequent product separation and extraction process. Our results confirmed that staged-addition glucoamylase strategy was feasible to effective production of CA.

Effects of mixed carbon sources on galactose and mannose content of exopolysaccharides and related enzyme activities in Ganoderma lucidum

This work investigated the effects of mixed carbon sources on the monosaccharide composition of exopolysaccharides (EPS) and activity of related enzymes, phosphoglucose isomerase (PGI), α-phosphoglucomutase (PGM), phosphomannose isomerase (PMI) and GDP-D-Man pyrophosphorylase (GMP), in the EPS biosynthesis by Ganoderma lucidum. Combinations of two carbon sources, glucose (Glc) + mannose (Man) and Glc + galactose (Gal), were applied at various mass ratios in the liquid culture of G. lucidum. The results showed that Glc, Gal, and Man were the major monosaccharides in G. lucidum EPS, and the combined mole percentages of monosaccharides were >80%, the same as those in the carbon source mixture. Activities of PGI, PMI, and GMP were correlated with Man mole percentage in EPSs and were enhanced by increasing the proportion of Gal for the carbon source Glc + Gal. PGM activity was correlated with Gal mole percentage and was reduced by increasing the Man proportion for Glc + Man. The expression of four genes encoding PGI, PMI, GMP and PGM reached peak levels on day 6 of culture, whereas the enzyme activities increased steadily from day 3 to 8. The monosaccharide composition of G. lucidum EPS for various mixed carbon source conditions thus appeared to be controlled by the translational level of genes encoding PGM, PGI, PMI, and GMP. These findings will be helpful to control the monosaccharide composition for desired biological activity of EPS produced by G. lucidum fermentation.

Bioactive Mushroom Polysaccharides: A Review on Monosaccharide Composition, Biosynthesis and Regulation

Mushrooms are widely distributed around the world and are heavily consumed because of their nutritional value and medicinal properties. Polysaccharides (PSs) are an important component of mushrooms, a major factor in their bioactive properties, and have been intensively studied during the past two decades. Monosaccharide composition/combinations are important determinants of PS bioactivities. This review summarizes: (i) monosaccharide composition/combinations in various mushroom PSs, and their relationships with PS bioactivities; (ii) possible biosynthetic pathways of mushroom PSs and effects of key enzymes on monosaccharide composition; (iii) regulation strategies in PS biosynthesis, and prospects for controllable biosynthesis of PSs with enhanced bioactivities.

Development of an inducible secretory expression system in Bacillus licheniformis based on an engineered xylose operon

The xylose operon can be an efficient biological component for regulatory expression uses in Bacillus licheniformis. However, its characteristic susceptibility to carbon catabolite repression (CCR) makes its application inconvenient. In this study, plasmids harboring the wild-type operons from three Bacillus species were constructed and introduced into B. licheniformis. These plasmids ensured secretory expression of maltogenic α-amylase (BLMA) in B. licheniformis under strict regulation. The glucose-mediated CCR was then alleviated by engineering the xylose operon of the expression system. Evidence showed that mutations in the highly conserved nucleotides of the identified catabolite responsive element (cre) consensus sequence prevented association of the regulator CcpA with DNA, thus resulting in an increase in BLMA activity of up to 12-fold. Furthermore, features of this engineered system for inducible expression were investigated. Induction in mid-log phase using 10 g/L xylose at 37 °C was found to be beneficial for promoting the accumulation of recombinant product, and the maximum yield of BlmMA reached 715.4 U/mL. This study contributes to the industrial application of the generally recognized as safe (GRAS) workhorse B. licheniformis.

β-Carotene from Yeasts Enhances Laccase Production of Pleurotus eryngii var. ferulae in Co-culture

Laccase is widely used in several industrial applications and co-culture is a common method for enhancing laccase production in submerged fermentation. In this study, the co-culture of four yeasts with Pleurotus eryngii var. ferulae was found to enhance laccase production. An analysis of sterilization temperatures and extraction conditions revealed that the stimulatory compound in yeasts was temperature-sensitive, and that it was fat-soluble. An LC-MS analysis revealed that the possible stimulatory compound for laccase production in the four yeast extracts was β-carotene. Moreover, the addition of 4 mg β-carotene to 150 mL of P. eryngii var. ferulae culture broth improved laccase production by 2.2-fold compared with the control (i.e., a monoculture), and was similar to laccase production in co-culture. In addition, the enhanced laccase production was accompanied by an increase of lac gene transcription, which was 6.2-time higher than the control on the fifth day. Therefore, it was concluded that β-carotene from the co-cultured yeasts enhanced laccase production in P. eryngii var. ferulae, and strains that produce β-carotene could be selected to enhance fungal laccase production in a co-culture. Alternatively, β-carotene or crude extracts of β-carotene could be used to induce high laccase production in large scale.