Hybrid scaffold bearing polymer-siloxane Schiff base linkage for bone tissue engineering.

Scaffolds that can provide the requisite biological cues for the fast regeneration of bone are highly relevant to the advances in tissue engineering and regenerative medicine. In the present article, we report the fabrication of a chitosan-gelatin-siloxane scaffold bearing interpolymer-siloxane Schiff base linkage, through a single-step dialdehyde cross-linking and freeze-drying method using 3-aminopropyltriethoxysilane as the siloxane precursor. Swelling of the scaffolds in phosphate buffered saline indicates enhancement with increase in siloxane concentration, whereas compressive moduli of the wet scaffolds reveal inverse dependence, owing to the presence of siloxane, rich in silanol groups. It is suggested that through the strategy of dialdehyde cross-linking, a limiting siloxane loading of 20 wt.% into a chitosan -gelatin matrix should be considered ideal for bone tissue engineering, because the scaffold made with 30 wt.% siloxane loading degrades by 48 wt.%, in 21 days. The hybrid scaffolds bearing Schiff base linkage between the polymer and siloxane, unlike the stable linkages in earlier reports, are expected to give a faster release of siloxanes and enhancement in osteogenesis. This is verified by the in vitro evaluation of the hybrid scaffolds using rabbit adipose mesenchymal stem cells, which revealed osteogenic cell-clusters on a polymer-siloxane scaffold, enhanced alkaline phosphatase activity and the expression of bone-specific genes, whereas the control scaffold without siloxane supported more of cell-proliferation than differentiation. A siloxane concentration dependent enhancement in osteogenic differentiation is also observed.

Biochemical characterization of raw-starch-digesting alpha amylase purified from Bacillus amyloliquefaciens.

Alpha amylase (E.C. 3.2.1.1) of Bacillus amyloliquefaciens produced by submerged fermentation was purified to near homogeneity by ion exchange chromatography. Through the process 38.6-fold increase in purity with a specific activity of 72 U/mg proteins was obtained. The apparent molecular weight of the purified enzyme was found to be 58 kDa by SDS-PAGE. The enzyme was relatively stable between pH 5.0-8.0 and temperature between 50 and 60 degrees C. The enzyme did not show any obligate requirement of metal ions but Ca2+ and Cu2+ enhanced the enzyme activity marginally and the thermostability was enhanced in the presence of Ca2+ ions. The purified enzyme exhibited maximal substrate specificity for amylose and efficiency in digesting various raw starches. The K(m) and V(max) of the enzyme was determined using both amylose and soluble starch as substrate. The analysis of the hydrolyzed products of soluble starch by thin layer chromatography showed the yield of maltosaccharides after 6 h of hydrolysis.

Genome shuffling of Lactobacillus delbrueckii mutant and Bacillus amyloliquefaciens through protoplasmic fusion for L-lactic acid production from starchy wastes.

Current study was focused on the development of a non-fastidious lactic acid producing strain having better growth rate, low pH tolerance and good productivity by genome shuffling of a mutant strain of Lactobacillus delbrueckii NCIM 2025 and an amylase producing non-fastidious Bacillus amyloliquefaciens ATCC 23842. After the third cycle of the protoplast fusion, lactic acid production by few fusants was monitored and the best fusant was selected for further studies. Optimization of the important process parameters for lactic acid production was conducted using Plackett-Burman design and response surface methodology. Selected fusant could utilize the liquefied cassava bagasse starch directly with minimum nutrient supplementation for lactic acid production. During validation, 40g/L of lactic acid was obtained ( approximately 96% conversion of starch to lactic acid) by using fusant inoculum (3%, v/v) from 83g/L cassava bagasse (starch content 50% w/w) supplemented with yeast extract and peptone (0.2% each, w/v) and the buffering agent (2% CaCO3, w/v).

Response surface methodology for the optimization of alpha amylase production by Bacillus amyloliquefaciens.

The aim of this work was to optimize the cultural and production parameters through the statistical approach for the synthesis of alpha amylase by Bacillus amyloliquefaciens in submerged fermentation (SmF) using a combination of wheat bran and groundnut oil cake (1:1) as the substrate. The process parameters influencing the enzyme production were identified using Plackett-Burman design. Among the various variables screened, the substrate concentration, incubation period and CaCl2 concentration were most significant. The optimum levels of these significant parameters were determined employing the response surface Box-Behnken design, which revealed these as follows: substrate concentration (12.5%), incubation period (42 h) and CaCl2 (0.0275 M).