A new extracellular glutaminase and urease-free L-asparaginase from Meyerozyma guilliermondii.

L-Asparaginase (L-ASNase) is a potent chemotherapeutic drug employed to treat leukemia and lymphoma. Currently, L-ASNases for therapeutic use are obtained from Escherichia coli and Dickeya chrysanthemi (Erwinia chrysanthemi). Despite their therapeutic potential, enzymes from bacteria are subject to inducing immune responses, resulting in a higher number of side effects. Eukaryote producers, such as fungi, may provide therapeutic alternatives through enzymes that induce relatively less toxicity and immune responses. Additional expected benefits from yeast-derived enzymes include higher activity and stability in physiological conditions. This work describes the new potential therapeutic candidate L-ASNase from the yeast Meyerozyma guilliermondii. A statistical approach (full factorial central composite design) was used to optimize L-ASNase production, considering L-asparagine and glucose concentration, pH of the medium, and cultivation time as independent factors. In addition, the crude enzymes were biochemically characterized, in terms of temperature and optimal pH, thermostability, pH stability, and associated glutaminase or urease activities. Our results showed that enzyme production increased after supplementing a pH 4.0 medium with 1.0% L-asparagine and 0.5% glucose during 75 h of cultivation. Under these optimized conditions, L-ASNase production reached 26.01 U mL-1, which is suitable for scale-up studies. The produced L-ASNase exhibits maximal activity at 37 °C and pH 7.0 and is highly stable under physiological conditions. In addition, M. guilliermondii L-ASNase has no associated glutaminase or urease activities, demonstrating its potential as a promising antineoplastic agent.

Efficient Conversion of Agroindustrial Waste into D(-) Lactic Acid by Lactobacillus delbrueckii Using Fed-Batch Fermentation.

PURPOSE: The goal of this paper is to describe the green conversion of agricultural waste products, such as molasses and corn steep liquor, into large amounts of D(-) lactic acid using a facilitated multipulse fed-batch strategy and affordable pH neutralizer. This is a very low-cost process because there is no need for hydrolysis of the waste products. The fed-batch strategy increases lactic acid productivity by avoiding inhibition caused by a high initial substrate concentration, and the selected controlling agent prevents cell stress that could be caused by high osmotic pressure of the culture media. METHODS: The effects of different carbon and nitrogen sources on lactic acid production were investigated, and the best concentrations of the medium components were determined. To optimize the culture conditions of the Lactobacillus delbrueckii strain, the effects of pH control, temperature, neutralizing agent, agitation, and inoculum size in batch cultures were investigated. Fed-batch strategies were also studied to improve production and productivity. RESULT: A high titer of D(-) lactic acid (162g/liter) was achieved after 48 hours of fermentation. Productivity at this point was 3.37 g/L·h. The optimum conditions were a temperature of 39°C, pH 5.5 controlled by the addition of Ca(OH)2, agitation at 150 rpm, and inoculum size of 25% (v/v). CONCLUSION: The production of high optical purity D(-) lactic acid through L. delbrueckii fermentation with molasses and corn steep liquor is a promising economical alternative process that can be performed on the industrial scale.

High-titer and productivity of l-(+)-lactic acid using exponential fed-batch fermentation with Bacillus coagulans arr4, a new thermotolerant bacterial strain.

Bacillus coagulans arr4 is a thermotolerant microorganism with great biotechnological potential for l-(+)-lactic acid production from granulated sugar and yeast extract. The highest l-(+)-lactic acid production was obtained with Ca(OH)2. The maximum production of l-(+)-lactic acid (206.81 g/L) was observed in exponential feeding using granulated sugar solution (900 g/L) and yeast extract (1%) at 50 °C, pH 6.5, and initial granulated sugar concentration of 100 g/L at 39 h. 5.3 g/L h productivity and 97% yield were observed, and no sugar remained. Comparing the simple batch with exponential fed-batch fermentation, the l(+) lactic acid production was improved in 133.22% and dry cell weight was improved in 83.29%, using granulated sugar and yeast extract. This study presents the highest productivity of lactic acid ever observed in the literature, on the fermentation of thermotolerant Bacillus sp. as well as an innovative and high-efficiency purification technology, using low-cost substances as Celite and charcoal. The recovery of lactic acid was 86%, with 100% protein removal, and the fermentation medium (brown color) became a colorless solution.

Production, purification, and characterization of a major Penicillium glabrum xylanase using Brewer's spent grain as substrate.

In recent decades, xylanases have been used in many processing industries. This study describes the xylanase production by Penicillium glabrum using brewer's spent grain as substrate. Additionally, this is the first work that reports the purification and characterization of a xylanase using this agroindustrial waste. Optimal production was obtained when P. glabrum was grown in liquid medium in pH 5.5, at 25 °C, under stationary condition for six days. The xylanase from P. glabrum was purified to homogeneity by a rapid and inexpensive procedure, using ammonium sulfate fractionation and molecular exclusion chromatography. SDS-PAGE analysis revealed one band with estimated molecular mass of 18.36 kDa. The optimum activity was observed at 60 °C, in pH 3.0. The enzyme was very stable at 50 °C, and high pH stability was verified from pH 2.5 to 5.0. The ion Mn(2+) and the reducing agents ß -mercaptoethanol and DTT enhanced xylanase activity, while the ions Hg(2+), Zn(2+), and Cu(2+) as well as the detergent SDS were strong inhibitors of the enzyme. The use of brewer's spent grain as substrate for xylanase production cannot only add value and decrease the amount of this waste but also reduce the xylanase production cost.