Mass production and characterization of an endoglucanase from Coleoptera insect (Monochamus saltuarius) in yeast Kluyveromyces lactis.

To harness the diverse industrial applications of cellulase, including its use in the food, pulp, textile, agriculture, and biofuel sectors, this study focused on the high-yield production of a bioactive insect-derived endoglucanase, Monochamus saltuarius GH Family 5 (MsGHF5). MsGHF5 was introduced into the genome of Kluyveromyces lactis to maintain expression stability, and mass production of the enzyme was induced using fed-batch fermentation. After 40 h of cultivation, recombinant MsGHF5 was successfully produced in the culture broth, with a yield of 29,000 U/L, upon galactose induction. The optimal conditions for the activity of purified MsGHF5 were determined to be a pH of 5 and a temperature of 35 °C, with the presence of ferrous ions enhancing the enzymatic activity by up to 1.5-fold. Notably, the activity of MsGHF5 produced in K. lactis was significantly higher than that produced in Escherichia coli, suggesting that glycosylation is crucial for the functional performance of the enzyme. This study highlights the potential use of K. lactis as a host for the production of bioactive MsGHF5, thus paving the way for its application in various industrial sectors.

Optimization of NaOH-catalyzed steam pretreatment of empty fruit bunch.

BACKGROUND: Empty fruit bunch (EFB) has many advantages, including its abundance, the fact that it does not require collection, and its year-round availability as a feedstock for bioethanol production. But before the significant costs incurred in ethanol production from lignocellulosic biomass can be reduced, an efficient sugar fractionation technology has to be developed. To that end, in the present study, an NaOH-catalyzed steam pretreatment process was applied in order to produce ethanol from EFB more efficiently. RESULTS: The EFB pretreatment conditions were optimized by application of certain pretreatment variables such as, the NaOH concentrations in the soaking step and, in the steam step, the temperature and time. The optimal conditions were determined by response surface methodology (RSM) to be 3% NaOH for soaking and 160°C, 11 min 20 sec for steam pretreatment. Under these conditions, the overall glucan recovery and enzymatic digestibility were both high: the glucan and xylan yields were 93% and 78%, respectively, and the enzymatic digestibility was 88.8% for 72 h using 40 FPU/g glucan. After simultaneous saccharification and fermentation (SSF), the maximum ethanol yield and concentration were 0.88 and 29.4 g/l respectively. CONCLUSIONS: Delignification (>85%) of EFB was an important factor in enzymatic hydrolysis using CTec2. NaOH-catalyzed steam pretreatment, which can remove lignin efficiently and requires only a short reaction time, was proven to be an effective pretreatment technology for EFB. The ethanol yield obtained by SSF, the key parameter determining the economics of ethanol, was 18% (w/w), equivalent to 88% of the theoretical maximum yield, which is a better result than have been reported in the relevant previous studies.

cDNA cloning and induction of tyrosine hydroxylase gene from the diamondback moth, Plutella xylostella.

We cloned a full-length tyrosine hydroxylase cDNA from the integument of the diamondback moth, Plutella xylostella. In the phylogenetic tree, tyrosine hydroxylase (PxTH) clustered with the other lepidopteran THs. Serine residues in the PxTH sequence, namely Ser(24), Ser(31), Ser(35), Ser(53), and Ser(65), were predicted to be the target sites for phosphorylation based on PROSITE analysis. In particular, Ser(35) of PxTH is highly conserved across a broad phylogenetic range of animal taxa including rat and human. Western blot analysis using both PxTH-Ab1 and PxTH-Ab2 polyclonal antibodies verified the expression of PxTH in all life cycle stages of P. xylostella, namely the larval, pupal, and adult stages. To examine the possible immune function of PxTH in P. xylostella, PxTH gene expression was investigated by RT-PCR and western blotting analysis after challenging P. xylostella with bacteria. PxTH expression was elevated 1 h post-infection and was continued till 12 h of post-infection relative to control larvae injected with sterile water.

Biodegradable intrascleral implant of triamcinolone acetonide in experimental uveitis.

PURPOSE: To evaluate the efficacy of a biodegradable intrascleral implant of triamcinolone acetonide (TA) in a rabbit model of experimental uveitis. METHODS: A disc-shaped TA implant (1 x 3 mm, containing 6.4 mg of TA), which is made by poly(D,L-lactide) (PLA) and TA, was intrasclerally implanted into eight albino rabbits after a subcutaneous injection of tuberculin antigen. Control animals (n = 8) received sham devices. Uveitis was induced with an intravitreal injection of tuberculin antigen. To induce chronic inflammation, all eyes were reinjected with intravitreal antigen on day 14 and were observed for 1 month. Aqueous protein concentrations and cell counts were determined on days 7, 14, and 28. Inflammation of the anterior chamber (AC) and vitreous opacity were graded clinically and retinal functions were evaluated electroretinographically and histologically. RESULTS: Anterior chamber cells, flare, and vitreous opacity scores showed significantly less inflammation in treated eyes than in untreated eyes (P < 0.05). Quantitative analyses of aqueous cell counts and protein concentrations showed significant decreases in treated eyes (P < 0.05). Histopathologic examinations showed less severe inflammation and tissue disorganization in treated eyes. CONCLUSIONS: The biodegradable intrascleral TA implant was found to be effective at suppressing the inflammation induced by experimental uveitis in rabbits for at least 4 weeks.

Comparison of the stability of postoperative alignment in sensory exotropia: adjustable versus non-adjustable surgery.

PURPOSE: To compare the success rates and stabilities of postoperative alignment between adjustable and the non-adjustable surgeries in the treatment of sensory exotropia. METHODS: A retrospective analysis was performed on all patients with sensory exotropia who had undergone unilateral lateral rectus recession and medial rectus resection (R&R) between January 1998 and August 2005. Thirty-four patients underwent conventional R&R, and 20 patients underwent R&R with adjustable suture of the lateral rectus. The surgical results between the two groups were analyzed with regard to the preoperative and post-operative deviation angles and the postoperative drift. The postoperative deviation angle was measured on postoperative day 1 as well as at two weeks, three months, six months and the final visit after surgery. RESULTS: There were no statistically significant differences in the mean preoperative and postoperative deviation angles between the two groups. In 30 (88%) patients in the non-adjustable group and 15 (75%) patients in the adjustable group, postoperative deviation was less than 15 prism diopters (PD) at the three month follow-up. There was no significant difference in the mean postoperative drift between the two groups. CONCLUSIONS: Strabismus surgery with adjustable sutures did not show a significantly better result than surgery without adjustable sutures in the treatment of sensory exotropia. Considering the amount of postoperative exodrift in both groups, we postulate that the immediate ocular alignment after surgery for sensory exotropia should be orthophoric or 5-6 PD of esodeviation.