An Effective Hybrid Strategy for Conversion of Biomass into Furfurylamine by Tandem Pretreatment and Biotransamination.

In this work, an effective hybrid strategy was developed for tandem conversion of biomass to furfurylamine with tin-based solid acid Sn-Maifanitum stone and recombinant Escherichia coli whole cells harboring ω-transaminase. 90.3 mM furfural was obtained from corncob (75 g/L) at 170 °C for 0.5 h over Sn-Maifanitum stone catalyst (3.5 wt%) in the aqueous media (pH 1.0), which could be further bioconverted into furfurylamine at 74.0% yield (based on biomass-derived furfural) within 20.5 h. Finally, an efficient recycling and reuse of Sn-Maifanitum stone catalyst and immobilized Escherichia coli AT2018 whole-cell biocatalyst was developed for the synthesis of furfurylamine from biomass in the one-pot reaction system.

Quantitative Analysis of the Substrate Specificity of Human Rhinovirus 3C Protease and Exploration of Its Substrate Recognition Mechanisms.

Human rhinovirus 3C protease (HRV 3C-P) is a high-value commercial cysteine protease that could specifically recognize the short peptide sequence of LEVLFQ↓GP. In here, a strategy based on our previous Yeast Endoplasmic Reticulum Sequestration Screening (YESS) approach was developed in Saccharomyces cerevisiae, a model microorganism, to fully characterize the substrate specificity of a typical human virus protease, HRV 3C-P, in a quantitative and fast manner. Our results demonstrated that HRV 3C-P had very high specificity at P1 and P1' positions, only recognizing Gln/Glu at the P1 position and Gly/Ala/Cys/Ser at the P1' position, respectively. Comparably, it exhibited efficient recognition of most residues at the P2' position, except Trp. Further biochemical characterization through site mutagenesis, enzyme structural modeling, and comparison with other 3C proteases indicated that the S1 pocket of HRV 3C-P was constituted by neutral and basic amino acids, in which His160 and Thr141 specifically interacted with Gln or Glu residues at the substrate P1 position. Additionally, the stringent S1' pocket determined its unique property of only accommodating residues without or with short side chains. Based on our characterization, LEVLFQ↓GM was identified as a more favorable substrate than the original LEVLFQ↓GP at high temperature, which might be caused by the conversion of random coils to ß-turns in HRV 3C-P along with the temperature increase. Our studies prompted a further understanding of the substrate specificity and recognition mechanism of HRV 3C-P. Besides, the YESS-PSSC combined with the enzyme modeling strategy in this study provides a general strategy for deciphering the substrate specificities of proteases.

Construction of a trifunctional cellulase and expression in Saccharomyces cerevisiae using a fusion protein.

BACKGROUND: Cellulose is the most important component of lignocellulose, and its degradation requires three different types of enzymes to act synergistically. There have been reports of single gene duality, but no gene has been described to have more than two functions. Cloning and expression of fusion cellulases containing more than two kinds of catalytic domains has not been reported thus far. RESULTS: We synthesized three different cellulase genes and linked the three catalytic domains with a (G4S)3 flexible linker. The trifunctional cellulase gene (BCE) containing three types of cellulase functions was constructed and expressed in S. cerevisiae successfully. The ß-glucosidase, the exoglucanase and the endoglucanase activity of the trifunctional cellulase BCE reached 16.80 IU/mg, 2.26 IU/mg and 20.67 IU/mg, which was 46.27, 6.73 and 46.20% higher than the activities of the ß-glucosidase BG, the endoglucanase CBH and the endoglucanase EG. The filter paper enzyme activity of BCE was higher than those of BG, CBH and EG, reached 2.04 IU/mg. CONCLUSIONS: The trifunctional cellulase BCE was designed based on ß-glucosidase BG, endoglucanase EG and exoglucanase CBH, and it possessed ß-glucosidase activity, endoglucanase activity and exoglucanase activity simultaneously. The BCE has better filter paper activity, it means the potential practical application.

Construction of recombinant Yarrowia lipolytica and its application in bio-transformation of lignocellulose.

Lignocellulose is a polysaccharide and an abundant biomass resource that widely exists in grains, beans, rice, and their by-products. Over 10 million tons of lignocellulose resources and processing products are produced every year in China. Three recombinant Y. lipolytica strains with cellulase (ß-glucosidase, endoglucanase and cellobiohydrolase) were constructed. The enzymatic activities of these enzymes were 14.181 U/mL, 16.307 U/mL, and 17.391 U/mL, respectively. The whole cell cellulases were used for a stover bio-transformation. The celluloses in the stover were partly degraded by the cellulases, and the degradation products were transformed into single cell protein (SCP) by the Y. lipolytica cells. After 15 d of fermentation with the whole cell cellulases, the protein content of the maize stover and the rice straw reached 16.23% and 14.75%, which increased by 168.26% and 161.52% compared with the control, respectively. This study provides a new stage for the efficient utilization of stover in the feed industry.

Synergistic effect of cellulase and xylanase during hydrolysis of natural lignocellulosic substrates.

Synergistic combination of cellulase and xylanase has been performed on pre-treated substrates in many previous studies, while few on natural substrates. In this study, three unpretreated lignocellulosic substrates were studied, including corncob, corn stover, and rice straw. The results indicated that when the mixed cellulase and xylanase were applied, reducing sugar concentrations were calculated as 19.53, 15.56, and 17.35mg/ml, respectively, based on the 3,5 dinitrosalicylic acid (DNS) method. Compared to the treatment with only cellulose, the hydrolysis yields caused by mixed cellulase and xylanase were improved by 133%, 164%, and 545%, respectively. In addition, the conversion yield of corncob, corn stover, and rice straw by cellulase-xylanase co-treatment reached 43.9%, 48.5%, and 40.2%, respectively, based on HPLC analysis, which confirmed the synergistic effect of cellulase-xylanase that was much higher than either of the single enzyme treatment. The substrate morphology was also evaluated to explore the synergistic mechanism of cellulase-xylanase.

Simultaneous saccharification and fermentation of corncobs with genetically modified Saccharomyces cerevisiae and characterization of their microstructure during hydrolysis.

Cellulose is an abundant natural polysaccharide that is universally distributed. It can be extracted from corncobs, which are inexpensive, easily accessible, renewable, and environmentally friendly. A common strategy for effectively utilizing cellulose is efficient heterogeneous expression of cellulase genes in Saccharomyces cerevisiae. However, the improvement of cellulose utilization is a relevant issue. Based on our previous findings, we constructed an integrated secretion expression vector, pHBM368-pgk, containing a constitutive promoter sequence. Three genetically modified S. cerevisiae strains containing heterologous ß-glucosidase, exoglucanase, and endoglucanase genes were constructed. The results of a 1-L bioreactor fermentation process revealed that the mixed recombinant S. cerevisiae could efficiently carry out simultaneous saccharification and fermentation (SSF) by using corncobs as the sole carbon source. The ethanol concentration reached 6.37 g/L after 96 hours of fermentation, which was about 3 times higher than that produced by genetically modified S. cerevisiae with the inducible promoter sequence. To investigate the microstructure characteristics of hydrolyzed corncobs during the fermentation process, corncob residues were detected by using a scanning electron microscope. This study provides a feasible method to improve the effect of SSF using corncobs as the sole carbon source.

Cell-surface display of the active mannanase in Yarrowia lipolytica with a novel surface-display system.

A novel surface-display system was constructed using the cell-wall anchor protein Flo1p from Saccharomyces cerevisiae, the mannanase (man1) from Bacillus subtilis fused with the C-terminus of Flo1p and the 6xHis tag was inserted between Flo1p and man1. The fusion protein was displayed on the cell surface of Yarrowia lipolytica successfully, and it was confirmed by immunofluorescence. In succession, the surface-displayed mannanase was characterized. The optimum catalytic conditions for the recombinant mannanase were 55 degrees C at pH 6.0, and it exhibited high stability against pH variation. The highest activity of the recombinant mannanase reached 62.3 IU/g (dry cell weight) after the recombinant was cultivated for 96 h in YPD medium [1% (w/v) yeast extract/2% (w/v) peptone/2% (w/v) glucose]. To our knowledge, the present paper is the first to report that high-activity mannanase is displayed on the cell surface of Y. lipolytica with Flo1p.

Cell surface display of functionally active lipases from Yarrowia lipolytica in Pichia pastoris.

The lipase genes of Yarrowia lipolytica, LIPY7 and LIPY8, fused with FLO-flocculation domain sequence from Saccharomyces cerevisiae at their N-termini, were expressed in Pichia pastoris KM71. Following the induction with methanol, the recombinant proteins were displayed on the cell surface of P. pastoris, as confirmed by the confocal laser scanning microscopy. The LipY7p and LipY8p were anchored on P. pastoris via the flocculation functional domain of Flo1p. The surface-displayed lipases were characterized for their application as the whole-cell biocatalyst. These lipases can also be cleaved off from their anchor by enterokinase treatment to yield functionally active proteins in the supernatant offering an alternative purification method for LipY7p and LipY8p.

Expression and purification of two lipases from Yarrowia lipolytica AS 2.1216.

We isolated two lipase genes LIPY7, LIPY8 from Yarrowia lipolytica CGMCC (China general microbiological culture collection center) AS 2.1216. The LIPY7 and LIPY8 genes encode a 366 and a 371-amino acid protein, respectively. The lipase genes with 6 x His tag sequence were cloned into expression vector pPIC9K and successfully integrated into a heterologous fungal host Pichia pastoris KM71, respectively. The recombinants were induced by methanol to secrete active lipases into cultural medium. The recombinant lipases were also purified and characterized.

[Secreted expression of Bacillus pumilus xylanase gene in Pichia pastoris and study on enzymatic properties].

The endo-1,4-xylanase gene from Bacillus pumilus HB030 was cloned into the Pichia pastoris expression vector, pPIC9k, the recombinant plasmid was named pHBM220. The digested recombinant plasmid pHBM220 was transformed into Pichia pastoris KM71, GS115, SMD1168, respectively. The recombinant Pichia pastoris KM71 (pHBM220), GS115 (pHBM220), SMD1168 (pHBM220) secreted functional endo-1,4-xylanase, and the enzymatic activities reached 10.80IU/mL, 11.63IU/mL, 9.68IU/mL, respectively. The temperature and pH optimum for the recombinant xylanase were 60 degrees C and pH5.5, respectively.