Purification and Characterization of Strong Simultaneous Enzyme Production of Protease and α-Amylase from an Extremophile-Bacillus sp. FW2 and Its Possibility in Food Waste Degradation

Microbial enzymes such as protease and amylase are valuable enzymes with various applications, widely investigated for their applications in degradation of organic waste, biofuel industries, agricultural, pharmaceuticals, chemistry, and biotechnology. In particular, extremophiles play an important role in biorefinery due to their novel metabolic products such as high value catalytic enzymes that are active even under harsh environmental conditions. Due to their potentials and very broad activities, this study isolated, investigated, and characterized the protease- and amylase-producing bacterial strain FW2 that was isolated from food waste. Strain FW2 belongs to the genus Bacillus and was found to be closest to Bacillus amyloliquefaciens DSM 7T with a similarity of 99.86%. This strain was able to degrade organic compounds at temperatures from −6 °C to 75 °C (but weak at 80 °C) under a wide pH range (4.5–12) and high-salinity conditions up to 35% NaCl. Maximum enzyme production was obtained at 1200 ± 23.4 U/mL for protease and 2400 ± 45.8 U/mL for amylase for 4 days at pH 7–7.5, 40–45 °C, and 0–10% NaCl. SDS-PAGE analysis showed that the molecular weights of purified protease were 28 kDa and 44 kDa, corresponding to alkaline protease (AprM) and neutral protease (NprM), respectively, and molecular weight of α-amylase was 55 kDa. Degradation food waste was determined after 15 days, observing a 69% of volume decrease. A potential commercial extremozyme-producing bacteria such as strain FW2 may be a promising contributor to waste degradation under extreme environmental conditions.

Cellulase Production by Penicillium Oxalicum Ti-11 with Traditional Chinese Medicine Residue as Substrate

PurposeThe study aims to search for efficient cellulase producer and explore the possibility of traditional Chinese medicine residue as a substrate for cellulase production, so as to realize the waste utilization of traditional Chinese medicine residue.MethodsThe cellulase-producing strain was identified through morphological and molecular biological methods. Scanning electron microscope (SEM) and Fourier transform infrared spectroscopy (FTIR) were used to characterize the structure of traditional Chinese medicine residues before and after fermentation. The enzyme activity was determined by DNS method, and the enzyme production conditions were optimized by single factor and response surface methodology.ResultThe strain grew well in forsythia leaf residue, and the highest FPA could reach 2.06 IU/mL. In addition, the structural characteristics of traditional Chinese medicine residue that before and after enzymatic hydrolysis were characterized by SEM and FTIR. The results showed that the structure of the residue was destroyed after enzymatic hydrolysis, the damage of forsythia leaf residue was the most serious, and enzymatic hydrolysis promoted the dissolution of cellulose, lignin and hemicellulose. The enzyme production conditions of the strain were optimized by Plackett-Burman design and response surface analysis. The FPA could reach 2.79 IU/mL under the optimal conditions of FLR concentration 24.84 g/L, (NH4)2SO4 concentration 2 g/L, temperature 34.44℃, pH 6.20, rotational speed 200rpm, inoculum 6%, which was 35.44% higher than that before optimization.ConclusionsThe results showed that traditional Chinese medicine residue could be used as the induced substrate for fungal cellulase production. This study provides an idea for the low-cost production of fungal cellulase and the waste utilization of traditional Chinese medicine residue.

Metabolic engineering of Bacillus subtilis with an endopolygalacturonase gene isolated from Pectobacterium. carotovorum; a plant pathogenic bacterial strain

Pectinolytic enzymes or pectinases are synthesized naturally by numerous microbes and plants. These enzymes degrade various kinds of pectin which exist as the major component of the cell wall in plants. A pectinase gene encoding endo-polygalacturonase (endo-PGase) enzyme was isolated from Pectobacterium carotovorum a plant pathogenic strain of bacteria and successfully cloned into a secretion vector pHT43 having σA-dependent promoter for heterologous expression in Bacillus subtilis (WB800N).The desired PCR product was 1209bp which encoded an open reading frame of 402 amino acids. Recombinant proteins showed an estimated molecular weight of 48 kDa confirmed by sodium dodecyl sulphate–polyacrylamide-gel electrophoresis. Transformed B. subtilis competent cells harbouring the engineered pHT43 vector with the foreign endo-PGase gene were cultured in 2X-yeast extract tryptone medium and subsequently screened for enzyme activity at various temperatures and pH ranges. Optimal activity of recombinant endo-PGase was found at 40°C and pH 5.0. To assay the catalytic effect of metal ions, the recombinant enzyme was incubated with 1 mM concentration of various metal ions. Potassium chloride increased the enzyme activity while EDTA, Zn++ and Ca++, strongly inhibited the activity. The chromatographic analysis of enzymatic hydrolysates of polygalacturonic acid (PGA) and pectin substrates using HPLC and TLC revealed tri and tetra-galacturonates as the end products of recombinant endo-PGase hydrolysis. Conclusively, endo-PGase gene from the plant pathogenic strain was successfully expressed in Bacillus subtilis for the first time using pHT43 expression vector and could be assessed for enzyme production using a very simple medium with IPTG induction. These findings proposed that the Bacillus expression system might be safer to escape endotoxins for commercial enzyme production as compared to yeast and fungi. Additionally, the hydrolysis products generated by the recombinant endo-PGase activity offer their useful applications in food and beverage industry for quality products.