Enzyme cascade converting cyclohexanol into ε-caprolactone coupled with NADPH recycling using surface displayed alcohol dehydrogenase and cyclohexanone monooxygenase on E. coli

The application of enzymes as biocatalysts in industrial processes has great potential due to their outstanding stereo-, regio- and chemoselectivity. Using autodisplay, enzymes can be immobilized on the cell surface of Gram-negative bacteria such as Escherichia coli. In the present study, the surface display of an alcohol dehydrogenase (ADH) and a cyclohexanone monooxygenase (CHMO) on E. coli was investigated. Displaying these enzymes on the surface of E. coli resulted in whole-cell biocatalysts accessible for substrates without further purification. An apparent maximal reaction velocity V_MAX(app) for the oxidation of cyclohexanol with the ADH whole-cell biocatalysts was determined as 59.9 mU ml⁻¹. For the oxidation of cyclohexanone with the CHMO whole-cell biocatalysts a V_MAX(app) of 491 mU ml⁻¹ was obtained. A direct conversion of cyclohexanol to ε-caprolactone, which is a known building block for the valuable biodegradable polymer polycaprolactone, was possible by combining the two whole-cell biocatalysts. Gas chromatography was applied to quantify the yield of ε-caprolactone. 1.12 mM ε-caprolactone was produced using ADH and CHMO displaying whole-cell biocatalysts in a ratio of 1:5 after 4 h in a cell suspension of OD_578nm 10. Furthermore, the reaction cascade as applied provided a self-sufficient regeneration of NADPH for CHMO by the ADH whole-cell biocatalyst.     

A semi-synthetic diet for rearing Dipha aphidivora (Lepidoptera: Pyralidae), a promising predator of woolly aphid in sugarcane

Dipha aphidivora (Meyrick), a lepidopteran predator of sugarcane woolly aphid (SWA) Ceratovacuna lanigera Zehntner was successfully reared for three successive generations, for the first time on a freeze-dried beef liver-based larval semi-synthetic diet. We compared biological parameters viz., larval survival, adult weight, pre-and oviposition period, fertility, fecundity and female longevity of D. aphidivora reared on the semi-synthetic diet with the predators reared on SWA. Development time of larvae reared from first instar to pupation was 20.6 days on the semi-synthetic diet and 12 days on SWA, while survival of the larvae to adults was 61.8 and 91.8% on larval semi-synthetic diet and SWA, respectively. Fecundity recorded from semi-synthetic diet (41 eggs/female) was significantly less than those produced on SWA (58 eggs/female). However, fertility and longevity of the predators reared on SWA and semi-synthetic diet did not differ significantly. The study revealed the possibility of rearing D. aphidivora larvae using synthetic diet.     

Isolation and screening of <Emphasis Type="Italic">phlD</Emphasis><Superscript>+</Superscript> plant growth promoting rhizobacteria antagonistic to <Emphasis Type="Italic">Ralstonia solanacearum</Emphasis>

Tomato (Lycopersicon esculentum) is important widely grown vegetable in India and its productivity is affected by bacterial wilt disease infection caused by Ralstonia solanacearum. To prevent this disease infection a study was conducted to isolate and screen effective plant growth promoting rhizobacteria (PGPR) antagonistic to R. solanacearum. A total 297 antagonistic bacteria were isolated through dual culture inoculation technique, out of which forty-two antagonistic bacteria were found positive for phlD gene by PCR amplification using two primer sets Phl2a:Phl2b and B2BF:BPR4. The genetic diversity of phlD ⁺ bacteria was studied by amplified 16S rDNA restriction analysis and demonstrated eleven groups at 65% similarity level. Out of these 42 phlD ⁺ antagonistic isolates, twenty exhibited significantly fair plant growth promoting activities like phosphate solubilization (0.92–5.33%), 25 produced indole acetic acid (1.63–7.78 μg ml⁻¹) and few strains show production of antifungal metabolites (HCN and siderophore). The screening of PGPR (phlD ⁺) for suppression of bacterial wilt disease in glass house conditions was showed ten isolated phlD ⁺ bacteria were able to suppress infection of bacterial wilt disease in tomato plant (var. Arka vikas) in the presence R. solanacearum. The PGPR (phlD ⁺) isolates s188, s215 and s288 was observed to be effective plant growth promoter as it shows highest dry weight per plant (3.86, 3.85 and 3.69 g plant⁻¹ respectively). The complete absence of wilt disease symptoms in tomato crop plants was observed by these treatments compared to negative control. Therefore inoculation of tomato plant with phlD ⁺ isolate s188 and other similar biocontrol agents may prove to be a positive strategy for checking wilt disease and thus improving plant vigor.