Comparison of the hydrolysis of polyethylene terephthalate fibers by a hydrolase from Fusarium oxysporum LCH I and Fusarium solani f. sp. pisi

The hydrolysis of polyethylene terephthalate (PET) fibers by two fungal hydrolases was investigated. The hydrolase from a newly isolated Fusarium oxysporum strain (LCH 1) was more efficient in releasing terephthalic acid from PET fibers compared to the enzyme from F. solani f. sp. pisi DSM 62420 when equal amounts of p-nitrophenyl butyrate-hydrolyzing activity were employed. PET fabrics treated under the same conditions with the enzyme from F. oxysporum LCH 1 also showed a considerably higher increase in hydrophilicity compared to fabrics treated with the enzyme from F. solani f. sp. pisi DSM 62420.     

Tuning of the enzyme ratio in a neutral redox convergent cascade: A key approach for an efficient one-pot/two-step biocatalytic whole-cell system

The efficiency of a versatile in vivo cascade involving a promiscuous alcohol dehydrogenase, obtained from a biodiversity search, and a Baeyer–Villiger monooxygenase was enhanced by the independent control of the production level of each enzyme to produce ε-caprolactone and 3,4-dihydrocoumarin. This goal was achieved by adjusting the copy number per cell of Escherichia coli plasmids. We started from the observation that this number generally correlates with the amount of produced enzyme and demonstrated that an in vivo multi-enzymatic system can be improved by the judicious choice of plasmid, the lower activity of the enzyme that drives the limiting step being counter-balanced by a higher concentration. Using a preconception-free approach to the choice of the plasmid type, we observed positive and negative synergetic effects, sometimes unexpected and depending on the enzyme and plasmid combinations. Experimental optimization of the culture conditions allowed us to obtain the complete conversion of cyclohexanol (16 mM) and 1-indanol (7.5 mM) at a 0.5-L scale. The yield for the conversion of cyclohexanol was 80% (0.7 g ε-caprolactone, for the productivity of 244 mg·L ⁻¹·h ⁻¹) and that for 1-indanol 60% (0.3 g 3,4-dihydrocoumarin, for the productivity of 140 mg·L ⁻¹·h ⁻¹).     

Rearranging the domain order of a diabody-based IgG-like bispecific antibody enhances its antitumor activity and improves its degradation resistance and pharmacokinetics

One approach to creating more beneficial therapeutic antibodies is to develop bispecific antibodies (bsAbs), particularly IgG-like formats with tetravalency, which may provide several advantages such as multivalent binding to each target antigen. Although the effects of configuration and antibody-fragment type on the function of IgG-like bsAbs have been studied, there have been only a few detailed studies of the influence of the variable fragment domain order. Here, we prepared four types of hEx3-scDb-Fc, IgG-like bsAbs, built from a single-chain hEx3-Db (humanized bispecific diabody [bsDb] that targets epidermal growth factor receptor and CD3), to investigate the influence of domain order and fusion manner on the function of a bsDb with an Fc fusion format. Higher cytotoxicities were observed with hEx3-scDb-Fcs with a variable light domain (VL)–variable heavy domain (VH) order (hEx3-scDb-Fc-LHs) compared with a VH–VL order, indicating that differences in the Fc fusion manner do not affect bsDb activity. In addition, flow cytometry suggested that the higher cytotoxicities of hEx3-scDb-Fc-LH may be attributable to structural superiority in cross-linking. Interestingly, enhanced degradation resistance and prolonged in vivo half-life were also observed with hEx3-scDb-Fc-LH. hEx3-scDb-Fc-LH and its IgG2 variant exhibited intense in vivo antitumor effects, suggesting that Fc-mediated effector functions are dispensable for effective anti-tumor activities, which may cause fewer side effects. Our results show that merely rearranging the domain order of IgG-like bsAbs can enhance not only their antitumor activity, but also their degradation resistance and in vivo half-life, and that hEx3-scDb-Fc-LHs are potent candidates for next-generation therapeutic antibodies.     

Rearranging the domain order of a diabody-based IgG-like bispecific antibody enhances its antitumor activity and improves its degradation resistance and pharmacokinetics

One approach to creating more beneficial therapeutic antibodies is to develop bispecific antibodies (bsAbs), particularly IgG-like formats with tetravalency, which may provide several advantages such as multivalent binding to each target antigen. Although the effects of configuration and antibody-fragment type on the function of IgG-like bsAbs have been studied, there have been only a few detailed studies of the influence of the variable fragment domain order. Here, we prepared four types of hEx3-scDb-Fc, IgG-like bsAbs, built from a single-chain hEx3-Db (humanized bispecific diabody [bsDb] that targets epidermal growth factor receptor and CD3), to investigate the influence of domain order and fusion manner on the function of a bsDb with an Fc fusion format. Higher cytotoxicities were observed with hEx3-scDb-Fcs with a variable light domain (VL)–variable heavy domain (VH) order (hEx3-scDb-Fc-LHs) compared with a VH–VL order, indicating that differences in the Fc fusion manner do not affect bsDb activity. In addition, flow cytometry suggested that the higher cytotoxicities of hEx3-scDb-Fc-LH may be attributable to structural superiority in cross-linking. Interestingly, enhanced degradation resistance and prolonged in vivo half-life were also observed with hEx3-scDb-Fc-LH. hEx3-scDb-Fc-LH and its IgG2 variant exhibited intense in vivo antitumor effects, suggesting that Fc-mediated effector functions are dispensable for effective anti-tumor activities, which may cause fewer side effects. Our results show that merely rearranging the domain order of IgG-like bsAbs can enhance not only their antitumor activity, but also their degradation resistance and in vivo half-life, and that hEx3-scDb-Fc-LHs are potent candidates for next-generation therapeutic antibodies.     

Efficient biosynthesis of antitumor ganoderic acid HLDOA using a dual tunable system for optimizing the expression of CYP5150L8 and a Ganoderma P450 reductase

Ganoderic acid 3-hydroxy-lanosta-8,24-dien-26-oic acid (GA-HLDOA), an antitumor triterpenoid from the traditional Chinese medicinal higher fungus Ganoderma lucidum, is considered as a key precursor for biosynthesizing other ganoderic acids (GAs) with superior antitumor activities. Our previous study identified CYP5150L8 from G. lucidum as a lanosterol oxidase, and achieved heterologous biosynthesis of GA-HLDOA in Saccharomyces cerevisiae. However, low production of GA-HLDOA in either G. lucidum or heterologous host hindered its further investigation and application. In this study, we constructed a dual tunable system for balancing the expression of CYP5150L8 and a Ganoderma P450 reductase iGLCPR, and performed a comprehensive optimization of CYP5150L8 expression, iGLCPR expression, and glycerol usage. Then, we investigated the fermentation behavior of the best strain in optimized condition in flask and achieved 154.45 mg/L GA-HLDOA production, which was 10.7-fold higher compared with previous report. This study may facilitate the wide-spread application of GA-HLDOA and the discovery of unknown cytochrome P450s in downstream GAs biosynthesis.     

Purification and characterization of phenylacetate-coenzyme A ligase from a denitrifying Pseudomonas sp., an enzyme involved in the anaerobic degradation of phenylacetate

The enzyme catalysing the first step in the anaerobic degradation pathway of phenylacetate was purified from a denitrifying Pseudomonas strain KB 740. It catalyses the reaction phenylacetate+CoA+ATP phenylacetyl-CoA+AMP+PP_i and requires Mg²⁺. Phenylacetate-CoA ligase (AMP forming) was found in cells grown anaerobically with phenylacetate and nitrate. Maximal specific enzyme activity was 0.048 mol min^-1 x mg^-1 protein in the mid-exponential growth phase. After 640-fold purification with 18% yield, a specific activity of 24.4 mol min^-1 mg^-1 protein was achieved. The enzyme is a single polypeptide with Mr of 52 ±2 kDa. The purified enzyme shows high specificity towards the aromatic inducer substrate phenylacetate and uses ATP preferentially; Mn²⁺ can substitute for Mg²⁺. The apparent K _m values for phenylacetate, CoA, and ATP are 60, 150, and 290 M, respectively. The soluble enzyme has an optimum pH of 8.5, is insensitive to oxygen, but is rather labile and requires the presence of glycerol and/or phenylacetate for stabilization. The N-terminal amino acid sequence showed no homology to other reported CoA-ligases. The expression of the enzye was studied by immunodetection. It is present in cells grown anaerobically with phenylacetate, but not with mandelate, phenylglyoxylate, benzoate; small amounts were detected in cells grown aerobically with phenylacetate.