Isolation, identification and prokaryotic expression of a bacteriocin-like substance from Bacillus licheniformis / 生物工程学报

The ban on addition of antibiotics in animal feed in China has made the search for new antibiotics substitutes, e.g. bacteriocin, a hot topic in research. The present study successfully isolated an antibacterial substance producing strain of Bacillus sp. from alpaca feces by agar diffusion method, using Escherichia coli, Salmonella enterica, Staphylococcus aureus, Staphylococcus epidermidis, Micrococcus luteus and Listeria monocytogenes as indicator bacteria. The isolated strain was named as B. licheniformis SXAU06 based on colony morphology, Gram staining and 16S rRNA gene sequence. The antibacterial substance was isolated and purified through a series of procedures including (NH4)2SO4 precipitation, chloroform extraction, molecular interception and SDS-PAGE analysis. Bioinformatics analysis of the LC-MS/MS data indicated that the antibacterial substance was a bacteriocin-like substance (BLIS) with an approximate molecular weight of 14 kDa, and it was designated as BLIS_SXAU06. BLIS_SXAU06 exhibited high resistance to treatment of proteinase K, high temperature, high acidity and alkalinity. BLIS_SXAU06 was heterologously expressed in E. coli and the recombinant BLIS_SXAU06 exhibited effective antibacterial activity against S. aureus, S. epidermidis, M. luteus, and L. monocytogenes, showing potential to be investigated further.

Agroindustrial biomass for xylanase production by Penicillium chrysogenum: purification, biochemical properties and hydrolysis of hemicelluloses

Background: In this work, the xylanase production by Penicillium chrysogenum F-15 strain was investigated using agroindustrial biomass as substrate. The xylanase was purified, characterized and applied in hemicellulose hydrolysis. Results: The highest xylanase production was obtained when cultivation was carried out with sugar cane bagasse as carbon source, at pH 6.0 and 20°C, under static condition for 8 d. The enzyme was purified by a sequence of ion exchange and size exclusion chromatography, presenting final specific activity of 834.2 U·mg·prot-1. T he molecular mass of the purified enzyme estimated by SDS-PAGE was 22.1 kDa. The optimum activity was at pH 6.5 and 45°C. The enzyme was stable at 40°C with half-life of 35 min, and in the pH range from 4.5 to 10.0. The activity was increased in the presence of Mg+2 and Mn+2 and reducing agents such as DTT and ßmercaptoethanol, but it was reduced by Cu+2 and Pb+2 . The xylanase presented Km of 2.3 mM and Vmax of 731.8 U·mg·prot-1 with birchwood xylan as substrate. This xylanase presented differences in its properties when it was compared to the xylanases from other P. chrysogenum strains. Conclusion: The xylanase from P. chrysogenum F-15 showed lower enzymatic activity on commercial xylan than on hemicellulose from agroindustry biomass and its biochemistry characteristics, such as stability at 40°C and pH from 4.0 to 10.0, shows the potential of this enzyme for application in food, feed, pulp and paper industries and for bioethanol production.

A novel pH-stable, endoglucanase (JqCel5A) isolated from a salt-lake microorganism, Jonesia quinghaiensis

Background: Endoglucanase, one of three type cellulases, can randomly cleave internal p-1,4-linkages in cellulose polymers. Thus, it could be applied in agricultural and industrial processes. Results: A novel endoglucanase gene (JqCel5A) was cloned from Jonesia quinghaiensis and functionally expressed in Escherichia coli Rosetta (DE3). It contained 1722 bp and encoded a 573-residue polypeptide consisting of a catalytic domain of glycoside hydrolase family 5 (GH5) and a type 2 carbohydrate-binding module (CBM2), together with a predicted molecular mass of 61.79 kD. The purified JqCel5A displayed maximum activity at 55°C and pH 7.0, with 21.7 U/mg, 26.19 U/mg and 4.81 U/mg towards the substrate carboxymethyl cellulose, barley glucan and filter paper, respectively. Interestingly, JqCel5A exhibited high pH stability over a broad pH range of pH (3-11), and had good tolerance to a wide variety of deleterious chemicals including heavy metals and detergent. The catalytic mechanism of JqCel5A was also investigated by site mutagenesis and homology-modeling in this study. Conclusions: It was believed that these properties might make JqCel5A to be potentially used in the suitable industrial catalytic condition, which has a broad pH fluctuation and/or chemical disturbance.

Heterologous expression and characterization of Klebsiella oxytoca lysine decarboxylase / 生物工程学报

Cadaverine is a biogenic amine that has the potential to become an important platform chemical for the production of industrial polymers, such as polyamides and polyurethanes. We reported here a lysine decarboxylase from Klebsiella oxytoca. The lysine decarboxylase from Klebsiella oxytoca was cloned to Escherichia coli to get the strain LN18. The specific activity of the crude protein from LN18 reached 30 000 U. The molecular weight was about 80 kDa. The optimum temperature and pH of the crude protein were 55 ℃ and 5.5 respectively. The specific activity could keep over 30% at pH 8.0 compared the one at pH 5.5, much difference from Escherichia coli lysine decarboxylase CadA. Mg²⁺ was positive to the specific activity, whereas Fe²⁺, Zn²⁺ and Ca²⁺ were negative.

The influence of pH, polyethylene glycol and polyacrylic acid on the stability of stem bromelain

Enzyme stability is critical in biotechnology, pharmaceutical and cosmetic industries. Investigations on this subject have drawn attention because of its practical application. Bromelain is a thiol-endopeptidase, obtained from pineapple (Ananas comosus), known for its clinical and therapeutic applications, particularly to selective burn debridement and improvement of antibiotic action and anti-inflammatory activities. To date, the use of bromelain in pharmacological or industrial applications is limited, due to commercial availability, costs, and sensitivity to pH and temperature. Therefore, a better understanding of enzyme stability would be of great interest. The aim of this study was to evaluate bromelain activity and stability in several pH (2.0 to 8.0) and in polyethylene glycol and polyacrylic acid solutions. We observed that bromelain was able to maintain its stability at pH 5.0 for the temperatures studied. PEG solutions increased bromelain stability, but PAA solutions had the opposite effect.

Estabilidade de enzimas é uma questão fundamental em indústrias biotecnológicas, farmacêuticas e cosméticas. As investigações sobre o assunto têm chamado a atenção por sua aplicação prática. A bromelina é uma tiol-endopeptidase, obtida a partir do abacaxi (Ananas comosus). É conhecida por suas aplicações clínicas e terapêuticas, especialmente para desbridamento seletivo de queimaduras, melhoria de ações antibiótica e de atividades anti-inflamatórias. Até o momento, a utilização da bromelina em aplicações farmacológicas industriais é limitada, devido à disponibilidade comercial, os custos, a sensibilidade ao pH e temperatura. Portanto, a maior compreensão da estabilidade desta enzima seria de grande interesse. O objetivo deste estudo foi avaliar a estabilidade da atividade da bromelina em vários pH (2,0 a 8,0) e em soluções de polietilenoglicol e de ácido poliacrílico. Observamos que a bromelina foi capaz de manter a sua estabilidade em pH 5.0, em todas as temperaturas estudadas. Soluções de PEG aumentaram a estabilidade da bromelina, enquanto que soluções de PAA obtiveram efeito oposto.