Peizhou Yang

Effect of Alkali and Laccase Pretreatment of Brassica Campestris Straw: Architecture, Crystallisation, and Saccharification

The morphological architecture and enzymatic hydrolysis of Brassica campestris straw after pretreatment with alkali and then with laccase was investigated. The results show that the diameters and depths of holes on the surface using the alkali and laccase pretreatment are larger than those observed with alkali pretreatment alone. At the same time, the crystallinity indices after alkali and laccase pretreatment are 1.1% higher compared with those observed after alkali pretreatment alone. The components were investigated, and saccharification analysis was carried out. The results showed that the contents of glucose (121 ± 1.1 g/L), xylose (55.9 ± 1.4 g/L), and total reducing sugar (195.3 ± 8.2 g/L) with the alkali and laccase treatment are 38, 193, and 69% higher than with the alkali pretreatment alone. Correspondingly, saccharification reaches 48.8 and 28.9% respectively with the alkali and laccase pretreatment and with the alkali treatment alone.

Improvement of the activity and thermostability of microbial transglutaminase by multiple-site mutagenesis

Abstract Microbial transglutaminase (MTG) is an enzyme widely used in the food industry. Mutiple-site mutagenesis of Streptomyces mobaraensis transglutaminase was performed in Escherichia coli. According to enzymatic assay and thermostability study, among three penta-site MTG mutants (DM01-03), DM01 exhibited the highest enzymatic activity of 55.7 ± 1.4 U/mg and longest half-life at 50 °C (418.2 min) and 60 °C (24.8 min).

Hydrolysis of by-product adenosine diphosphate from 3'-phosphoadenosine-5'-phosphosulfate preparation using Nudix hydrolase NudJ.

Abstract3′-Phosphoadenosine-5′-phosphosulfate (PAPS) is the obligate cosubstrate and source of the sulfonate group in the chemoenzymatic synthesis of heparin, a clinically used anticoagulant drug. Previously, we have developed a method to synthesize PAPS with Escherichia coli crude extracts, which include three overexpressed enzymes and a fourth unidentified protein. The unknown protein degrades adenosine diphosphate (ADP), the by-product of PAPS synthesis reaction. To further understand and control the process of in vitro enzymatic PAPS synthesis, we decide to identify the fourth protein and develop a defined method to synthesize PAPS using purified enzymes. Here, we show that the purified Nudix hydrolase NudJ degrades ADP at high efficiency and serves as the fourth enzyme in PAPS synthesis. Under the defined condition of PAPS synthesis, all of the 10-mM ADP is hydrolyzed to form adenosine monophosphate (AMP) in a 15-min reaction. ADP is a better substrate for NudJ than adenosine triphosphate (ATP). Most importantly, the purified NudJ does not cleave the product PAPS. The removal of ADP makes the PAPS peak more separable from other components in the chromatographic purification process. This developed enzymatic approach of PAPS production will contribute to the chemoenzymatic synthesis of heparin.

Metabolic control analysis of l -lactate synthesis pathway in Rhizopus oryzae As 3.2686

The relationship between the metabolic flux and the activities of the pyruvate branching enzymes of Rhizopus oryzae As 3.2686 during l-lactate fermentation was investigated using the perturbation method of aeration. The control coefficients for five enzymes, pyruvate dehydrogenase (PDH), pyruvate carboxylase (PC), pyruvate decarboxylase (PDC), lactate dehydrogenase (LDH), and alcohol dehydrogenase (ADH), were calculated. Our results indicated significant correlations between PDH and PC, PDC and LDH, PDC and ADH, LDH and ADH, and PDC and PC. It also appeared that PDH, PC, and LDH strongly controlled the l-lactate flux; PDH and ADH strongly controlled the ethanol flux; while PDH and PC strongly controlled the acetyl coenzyme A flux and the oxaloacetate flux. Further, the flux control coefficient curves indicated that the control of the system gradually transferred from PDC to PC during the steady state. Therefore, PC was the key rate-limiting enzyme that controls the flux distribution.

CRISPR-Cas9 Approach Constructing Cellulase sestc-Engineered Saccharomyces cerevisiae for the Production of Orange Peel Ethanol

The development of lignocellulosic bioethanol plays an important role in the substitution of petrochemical energy and high-value utilization of agricultural wastes. The safe and stable expression of cellulase gene sestc was achieved by applying the clustered regularly interspaced short palindromic repeats-Cas9 approach to the integration of sestc expression cassette containing Agaricus biporus glyceraldehyde-3-phosphate-dehydrogenase gene (gpd) promoter in the Saccharomyces cerevisiae chromosome. The target insertion site was found to be located in the S. cerevisiae hexokinase 2 by designing a gRNA expression vector. The recombinant SESTC protein exhibited a size of approximately 44 kDa in the engineered S. cerevisiae. By using orange peel as the fermentation substrate, the filter paper, endo-1,4-β-glucanase, exo-1,4-β-glucanase activities of the transformants were 1.06, 337.42, and 1.36 U/mL, which were 35.3-fold, 23.03-fold, and 17-fold higher than those from wild-type S. cerevisiae, respectively. After 6 h treatment, approximately 20 g/L glucose was obtained. Under anaerobic conditions the highest ethanol concentration reached 7.53 g/L after 48 h fermentation and was 37.7-fold higher than that of wild-type S. cerevisiae (0.2 g/L). The engineered strains may provide a valuable material for the development of lignocellulosic ethanol.

High Level of Cellulase and β-Glucosidase Production by T.reesei and T.koningii Combinations on Corn Straws of Alkali Pretreatment

In order to obtain high level of β-glucosidase and filter paper activity production, the screening of strain combinations and the characteristic of enzymes from co-culture were investigated. The results showed that both the β-glucosidase (0.347 U/ml) and FPA (3.21 U/ml) reached the highest level via co-culture of T.reesei and T.koningii with the corn straw of alkali pretreatment as fermentation substrate. After 72 h of fermentation, both two enzyme activities reached the highest level. And the optimal temperature and pH value were 55°C and 5.5, respectively. These two enzyme stability decreased rapidly when the temperatures exceeded 90°C. The conversion efficiency reached 0.652 g reducing sugar per g pretreated corn straw using the enzymes from these two fungi, which was 1.49-fold and 1.64-fold compared with those from T.reesei (0.439 g/g) and T.koningii (0.398 g/g). The present work demonstrated an effective way to develop an efficient system for the bioconversion of agricultural waste straws to sugar.