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Phenylketonuria (PKU), a disease resulting in the disability to degrade phenylalanine (Phe) is an inborn error with a 1 in 10,000 morbidity rate on average around the world which leads to neurotoxicity. As an potential alternative to a protein-restricted diet, oral intake of engineered probiotics degrading Phe inside the body is a promising treatment, currently at clinical stage II (Isabella, et al., 2018). However, limited transmembrane transport of Phe is a bottleneck to further improvement of the probiotic's activity. Here, we achieved simultaneous degradation of Phe both intracellularly and extracellularly by expressing genes encoding the Phe-metabolizing enzyme phenylalanine ammonia lyase (PAL) as an intracellularly free and a cell surface-immobilized enzyme in Escherichia coli Nissle 1917 (EcN) which overcomes the transportation problem. The metabolic engineering strategy was also combined with strengthening of Phe transportation, transportation of PAL-catalyzed trans-cinnamic acid and fixation of released ammonia. Administration of our final synthetic strain TYS8500 with PAL both displayed on the cell surface and expressed inside the cell to the Pah F263S PKU mouse model reduced blood Phe concentration by 44.4% compared to the control EcN, independent of dietary protein intake. TYS8500 shows great potential in future applications for PKU therapy., (© 2022. Science China Press and Springer-Verlag GmbH Germany, part of Springer Nature.)

L-asparaginase, a therapeutic involved in cancer therapy, from Bacillus tequilensis PV9W (ansA gene) was cloned and over expressed in Escherichia coli BL21 (DE3), achieved the aim of maximizing the yield of the recombinant enzyme (6.02 ± 1.77 IU/mL) within 12 h. The native L-asparaginase of B. tequilensis PV9W was encapsulated using solid lipid particles by hot lipid emulsion method, which is reported for first time in this study. Subsequently, the lipid encapsulated L-asparaginase (LPE) was characterized by SEM, UV-Vis spectroscopy, FT-IR, SDS-PAGE and its thermo stability was also analyzed by TGA. Further characterization of LPE revealed that enzyme was highly stable for 25 days when stored at 25 °C, showed high pH (9) tolerance and longer trypsin half-life (120 min). In addition, the cytotoxic ability of LPE on HeLa cells was highly enhanced compared to the native L-asparaginase from Bacillus tequilensis PV9W. Moreover, better kinetic velocity and lower K m values of LPE aided to detect L-asparagine in cell extracts by Differential Pulse Voltammetry (DPV) method. The LPE preparation also showed least immunogenic reaction when tested on normal macrophage cell lines. This LPE preparation might thus pave way for efficient drug delivery and enhancing the stability of L-asparaginase for its therapeutic applications.

The results of treatment of 77 patients, ageing 18-71 yrs old, for an acute paraproctitis in 2010-2014 yrs were analyzed. A preventive puncture-flush enzymosanation of purulent foci, using immobilized bacterial proteinases (imozimase), metrogyl P in conjunction with low-intensive laser irradiation have permitted to conduct the optimal preoperative preparation of patients, to improve their state, to reduce the local inflammatory reactions intensity significantly.

A new method for the immobilization of phospholipase A2 (PLA2) has been developed to enhance the activity retention of immobilized PLA2. When PLA2 from the venom of Agkistrodon piscivorus piscivorus was pretreated with 4-nitro-3-octanoyl-oxybenzoic acid to acylate epsilon-amino groups of two lysines (Lys-7 and Lys-10) and the resulting acylated enzyme was covalently coupled onto carbonyldiimidazole-activated cross-linked agarose beads, the immobilized acylated enzyme showed high retention of activity toward various aggregated phospholipids. Toward densely packed phospholipid bilayers, such as large unilamellar vesicles of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, the immobilized acylated A. p. piscivorus PLA2 was 25-fold more active than the soluble A. p. piscivorus PLA2. The general applicability of our immobilization protocol was demonstrated by the high retention of activity achieved for the immobilized acylated PLA2 from the venom of Naja naja naja. In particular, full activity retention of the immobilized acylated A. p. piscivorus PLA2 toward phospholipids on the surface of human low density lipoproteins suggests its potential usefulness in a newly developed PLA2-based therapy for hypercholesterolemia.

Pretreatment characteristics of 138 dogs with malignant lymphoma were analyzed to determine prognostic factors associated with outcome (ie, complete response rate, time to relapse after complete response, survival time). Dogs were all treated for 10 weeks, using a standard induction chemotherapy protocol, and were then given asparaginase weekly. Once the disease became progressive, second-line chemotherapy was instituted. Age, sex, weight, clinical stage, performance grade, immunophenotype, and malignancy grade assigned according to the National Cancer Institute's Working Formulation were not associated with complete response rate. However, malignancy grade assigned according to the Kiel classification was found to be associated with complete response rate; dogs with high-grade malignancies had a significantly higher complete response rate than did dogs with low-grade malignancies. By means of multivariate analysis, clinical stage and immunophenotype were found to be prognostic factors for time to relapse (among dogs that had had a complete response) and survival time. In addition, malignancy grade assigned according to the Kiel classification was found to be a prognostic factor for time to relapse; whereas, malignancy grade assigned according to the Working Formulation was determined to be a prognostic factor for survival time.

L-asparaginase is an enzyme that inhibits protein synthesis by the depletion of sources of L-asparagine, which is necessary for transformed lymphoid cells to proliferate. L-asparaginase is used in the treatment of childhood acute lymphoblastic leukemia. A problem with L-asparaginase therapy is the immunogenicity of the enzyme and the development of anaphylactic reactions. Canine lymphoma is a predominantly B-cell tumor with widespread disease; without treatment, dogs with lymphoma usually survive 1-2 months. Canine lymphoma will respond to L-asparaginase therapy. A randomized double-blind study evaluated a polyethylene glycol (PEG) conjugate L-asparaginase combined with chemotherapy (vincristine, cyclophosphamide, doxorubicin, and prednisone). Thirty-five dogs were randomized to the PEG L-asparaginase group, and 34 dogs were randomized to the native L-asparaginase group. Thirty dogs (85.7%) achieved a complete remission (CR) with a median time to relapse of 217 days, and 32 (94.1%) dogs in the native L-asparaginase group achieved a CR with a median time to relapse of 214 days (P greater than 0.05). The asparaginase was well tolerated in both groups. Two dogs in the native L-asparaginase group had severe allergic reactions, and one dog in the PEG asparaginase group had a generalized urticarial reaction after repeated injections. This study indicates that PEG L-asparaginase has equal therapeutic efficacy to native L-asparaginase.

Since streptokinase and urokinase became available for clinical use, numerous attempts have been made to improve these useful thrombolytic agents. To decrease its antigenicity, streptokinase has been fragmented or coupled to human plasminogen or polyethylene glycols. With a plasmin B chain-streptokinase complex a more potent agent was obtained. To prolong their half-life, streptokinase and urokinase were immobilized with water-soluble carriers. Coupling urokinase with fibrin-specific antibodies increases its thrombolytic efficacy, at least in vitro. The only thrombolytic agents with a relative fibrin specificity available for clinical purposes are tissue-type plasminogen activator and single chain urokinase-type plasminogen activator. Mutants and hybrids of these molecules are being constructed and may further improve their fibrin specificity and therapeutic potential.

Multitubular enzyme reactors with immobilized phenylalanine ammonia lyase were tested in vitro and in vivo for depletion of phenylalanine in circulating blood. Sustained reduction of phenylalanine was achieved in less than 30 minutes. A 50% decrease of phenylalanine was obtained with a 2-hour application of enzyme reactors and was maintained for more than 2 days. Similar enzyme reactors have therapeutic potential for temporary management of phenylketonuric patients when their circulating phenylalanine becomes exceedingly high because of infection, fever, or pregnancy.

Phenylalanine ammonia-lyase immobilized within semipermeable microcapsules has an assayed enzyme activity which is 20% +/- 4% of the enzyme in free solution. The Km for the immobilized enzyme remained the same as that of the free enzyme. The pH optimum also remained unchanged at pH 8.5 +/- 1.0. At the lower pH range, enzyme activity is higher for the immobilized enzyme. Daily oral administration of microencapsulated phenylalanine ammonia-lyase to phenylketonuric rats decreased the systemic phenylalanine level by 35 +/- 8% in 2 days (P less than 0.05) and by 75 +/- 8% in 7 days (P less than 0.001).