Jiaxing Xu

An ionic liquid tolerant cellulase derived from chemically polluted microhabitats and its application in in situ saccharification of rice straw

A cellulase-producing fungus was isolated from chemically polluted microhabitats by [Amim][Cl] enrichment and identified as Aspergillus fumigatus. The maximum activity of the cellulase in 30% (v/v) ionic liquids (ILs) was detected in [Emim][DMP], [Amim][Cl] and [Emim][MA] as 127%, 111% and 109%, respectively, of its activity in buffer, suggesting its superior performance in high concentration ILs. Strikingly, although its initial activity varied in each IL, its half-life was longer in most ILs than in buffer, evidence of a high conformational stability of the enzyme that is essential for maintaining the remaining activity in relevant media. It noteworthy that 1-3M NaCl can activate the cellulase somewhat. More gratifyingly, a compatible IL-cellulase system based on the cellulase was developed, and its use significantly improved the saccharification rate of rice straw from 53% to 88% versus the control, demonstrating its potential for efficient transformation of lignocellulose to glucose in a single-step process.

Advances in improving the performance of cellulase in ionic liquids for lignocellulose biorefinery

Ionic liquids (ILs) have been considered as a class of promising solvents that can dissolve lignocellulosic biomass and then provide enzymatic hydrolyzable holocellulose. However, most of available cellulases are completely or partially inactivated in the presence of even low concentrations of ILs. To more fully exploit the benefits of ILs to lignocellulose biorefinery, it is critical to improve the compatibility between cellulase and ILs. Various attempts have been made to screen natural IL-tolerant cellulases from different microhabitats. Several physical and chemical methods for stabilizing cellulases in ILs were also developed. Moreover, recent advances in protein engineering have greatly facilitated the rational engineering of cellulases by site-directed mutagenesis for the IL stability. This review is aimed to provide the first detailed overview of the current advances in improving the performance of cellulase in non-natural IL environments. New ideas from the most representative progresses and technical challenges will be summarized and discussed.

An organic solvent-stable protease from organic solvent-tolerant Bacillus cereus WQ9-2: purification, biochemical properties, and potential application in peptide synthesis.

An extracellular solvent-stable protease producing bacterium WQ9-2 was isolated and identified taxonomically as Bacillus cereus. The protease from strain WQ9-2 was purified to homogeneity with an estimated molecular mass of 37 kDa. The purified protease showed maximum activity at 50 °C and pH 8.0. The protease may be classified as a metalloprotease since it was strongly inhibited by EDTA and 1,10-phenanthroline. The protease showed extreme activity and stability in the presence of both 50% (v/v) hydrophilic or hydrophobic solvents. The synthesis of the precursor (Cbz-Ala-Phe-NH₂) of a bitter dipeptide could be catalyzed by the protease in the presence of 50% dimethylsulfoxide with the product crystals separating directly. The protease displayed broad catalysis specificity for carboxyl component and different substrate preferences in various solvent media, thus confirming its potential application in peptide synthesis.

A novel ionic liquid-tolerant Fusarium oxysporum BN secreting ionic liquid-stable cellulase: Consolidated bioprocessing of pretreated lignocellulose containing residual ionic liquid

In this study, microbial communities from chemicals polluted microhabitats were cultured with the addition of imidazolium-based ionic liquid (IL) to enrich for IL-tolerant microbes. A strain of Fusarium oxysporum BN producing cellulase from these enrichments was capable of growing in 10% (w/v) 1-ethyl-3-methylimidazolium phosphinate, much higher than the normal IL concentrations in the lignocellulose regenerated from ILs. Cellulase secreted by the strain showed high resistance to ILs based on phosphate and sulfate radicals, evidencing of a high conformational stability in relevant media. Gratifyingly, F. oxysporum BN can directly convert IL-pretreated rice straw to bioethanol via consolidated bioprocessing (I-CBP). At optimum fermentation condition, a maximum ethanol yield of 0.125 g ethanol g(-1) of rice straw was finally obtained, corresponding to 64.2% of the theoretical yield.

Efficient chemo-enzymatic synthesis of endomorphin-1 using organic solvent stable proteases to green the synthesis of the peptide

Endomorphin-1 (Tyr-Pro-Trp-Phe-NH2, EM-1), an effective analgesic, was efficiently synthesized by a combination of enzymatic and chemical methods. Peptide Boc-Trp-Phe-NH2 was synthesized with a high yield of 97.1% by the solvent-stable protease WQ9-2 in a 20% methanol medium. The maximum concentration (141 g L−1) of Boc-Trp-Phe-NH2 was obtained with an economical molar ratio of the substrate of 1 : 1. The products crystallized and separated from the substrates without purification, followed by the removal of the Boc group with trifluoroacetic acid to generate Trp-Phe-NH2. Using the efficient mixed carbonic anhydride method, Boc-Tyr-Pro-OH was synthesized chemically. The tetrapeptide Boc-Tyr-Pro-Trp-Phe-NH2 was synthesized with a yield of 84.5% by another organic solvent-tolerant protease, PT121, from Boc-Tyr-Pro-OH and Trp-Phe-NH2 in an organic–aqueous biphasic system and was extracted with ethyl acetate, shifting the equilibrium of the synthesis. EM-1 was obtained by removal of the Boc group from Boc-Tyr-Pro-Trp-Phe-NH2 in a high yield of 91% due to the free protection of the side-chain of the Tyr phenolic hydroxy group. After a one-step purification in the final step by using high speed countercurrent chromatography (HSCCC), EM-1 was obtained with a purity greater than 99.8%. The chemo-enzymatic synthesis of EM-1 proved to be efficient, productive, with minimal side-chain protection and simple purification, thus greening the synthesis of the peptide.

Enhancement in ionic liquid tolerance of cellulase immobilized on PEGylated graphene oxide nanosheets: Application in saccharification of lignocellulose.

The objective of the present work was to improve ionic liquid (IL) tolerance of cellulase based on the exploration of functional nanoscale carriers for potential application in lignocellulosic biorefinery. PEGylated graphene oxide (GO) composite was successfully fabricated by chemical binding of 4-arm-PEG-NH2 and GO and applied to the immobilization of cellulase. The PEGylated GO-Cellulase retained 61% of the initial activity in 25% (w/v) 1-butyl-3-methylimidazolium chloride ([Bmim][Cl]) while free cellulase only retained 2%. The IL stability was enhanced more than 30 times. The relatively minor change in Km value (from 2.7 to 3.2mgmL(-1)) after the immobilization suggested that PEGylated GO-Cellulase was capable of closely mimicking the performance of free enzyme. After treating rice straw with [Bmim][Cl] and dilution to a final IL concentration of 15% (w/v), the slurry was directly hydrolyzed using PEGylated GO-Cellulase without IL removing and a high hydrolysis rate of 87% was achieved.

Calcium-ion-induced stabilization of the protease from Bacillus cereus WQ9-2 in aqueous hydrophilic solvents: effect of calcium ion binding on the hydration shell and intramolecular interactions

The neutral protease WQ from Bacillus cereus is stable in various aqueous organic mixtures, with the exception of those containing acetonitrile (ACN) and dimethylformamide (DMF). The stability of the enzyme in aqueous hydrophilic solvents was dramatically enhanced with the addition of calcium ions, with the degree of improvement in the half-life relative to different solutions ranging from fourfold to more than 70-fold. Studies of the kinetic constants showed that calcium ions induced slight conformational changes in the active site of the enzyme in aqueous ACN. We investigated the molecular mechanisms underlying this stabilizing effect by employing a combination of biophysical techniques and molecular dynamics simulation. In aqueous ACN, the intrinsic fluorescence and circular dichroism analysis demonstrated that the addition of calcium ions induced a relatively compact conformation and maintained both the native-like microenvironment near the tryptophan residues and the secondary structure. Alternatively, homology modeling confirmed the location of four calcium-ion-binding sites in the enzyme, and molecular dynamics simulation revealed that three other calcium ions were bound to the surface of the enzyme. Calcium ions, known as a type of kosmotrope, can strongly bond with water molecules, thus aiding in the formation of the regional hydration shell required for the maintenance of enzyme activity. In addition, the introduction of calcium ions resulted in the formation of additional ionic interactions, providing propitious means for protein stabilization. Thus, the stronger intramolecular interactions were also expected to contribute partially to the enhanced stability of the enzyme in an aqueous organic solvent.

Production of poly(β-l-malic acid) by Aureobasidium pullulans HA-4D under solid-state fermentation

Poly(β-l-malic acid) (PMA) production by Aureobasidium pullulans HA-4D was carried out through solid-state fermentation (SSF) using agro-industrial residues. Maximum PMA production (75.4mg/g substrate) was obtained from a mixed substrate of sweet potato residue and wheat bran (1:1, w/w) supplemented with NaNO3 (0.8%, w/w) and CaCO3 (2%, w/w), with an initial moisture content of 70% and inoculum size of 13% (v/w) for 8days. Repeated-batch SSF was successfully conducted for 5 cycles with a high productivity. The scanning electron microscopy showed that the yeast-like cells of A. pullulans HA-4D could grow well on the solid substrate surface. Moreover, the cost analysis showed that the unit price of PMA in SSF was much lower than that of SmF. This is the first report on PMA production via SSF, and this study provided a new method to produce PMA from inexpensive agro-industrial residues.

Synergistic effects of metal salt and ionic liquid on the pretreatment of sugarcane bagasse for enhanced enzymatic hydrolysis

High cost of ionic liquids (ILs) restricts the industrial application of IL-mediated lignocellulose pretreatment. In this study, a simple and economic technology for the pretreatment of natural lignocellulose was developed. The delignification capacity of aqueous choline ornithine ([Cho][Orn]) and hemicellulose-removal capacity of metal salt FeCl2 were combined. The changes of morphological structure and composition indicated a synergistic interaction of [Cho][Orn] and FeCl2 in the pretreatment process. The delignification and hemicellulose-removal capacity of aqueous [Cho][Orn]50% solution was significantly improved in the presence of FeCl2 by 28% and 53%, respectively. The combination use of FeCl2 and [Cho][Orn] made it possible to save the amount of IL used for pretreatment in half. Enhancement effect of metal salts on the IL-pretreatment efficiency was proved.

Effects of osmotic pressure and pH on citric acid and erythritol production from waste cooking oil by Yarrowia lipolytica

Erythritol and citric acid could be produced from waste cooking oil (WCO) by Yarrowia lipolytica under different medium conditions, and osmotic pressure together with pH were considered to be the critical factors in this process. High osmotic pressure (2.76 osmol/L) combined with low pH (pH 3.0) promoted the highest yield of erythritol (21.8 g/L) accompanied by low‐producing citric acid (2.5 g/L). By contrast, the highest citric acid biosynthesis (12.6 g/L) was detected under a pH of 6.0 and an osmotic pressure of 0.75 osmol/L, when only 4.0 g/L of erythritol was yielded. Moreover, lipase activities in these two media were also detected, and pH 3.0–OP 2.76 was supposed to be more beneficial to lipase activity. Biochemical pathways involved in the biosynthesis of erythritol and citric acid were subsequently investigated, and the products yielded from WCO were assumed to be correlated with the activities of transketolase, erythrose reductase, citrate synthase, and glycerol kinase. However, RT‐PCR analysis revealed that mRNA levels of these enzymes did not significantly differ, confirming that metabolic flux regulations of erythritol and citric acid mostly took place at the post‐transcriptional level.