Inulooligosaccharides (IOS) represent an important class of oligosaccharides at industrial scale. An efficient conversion of inulin to IOS through endoinulinase from Aspergillus niger is presented. A 1482 bp codon optimized gene fragment encoding endoinulinase from A. niger DSM 2466 was cloned into pPIC9K vector and was transformed into Pichia pastoris KM71. Maximum activity of the recombinant endoinulinase, 858 U/mL, was obtained at 120 h of the high cell density fermentation process. The optimal conditions for inulin hydrolysis using the recombinant endoinulinase were investigated. IOS were harvested with a high concentration of 365.1 g/L and high yield up to 91.3%. IOS with different degrees of polymerization (DP, mainly DP 3–6) were distributed in the final reaction products.

Inulin is a readily available feedstock for cost-effective production of biochemicals. To date, several studies have explored the production of bioethanol, high-fructose syrup and fructooligosaccharide, but there are no studies regarding the production of d-lactic acid using inulin as a carbon source. In the present study, chicory-derived inulin was used for d-lactic acid biosynthesis by Lactobacillus bulgaricus CGMCC 1.6970. Compared with separate hydrolysis and fermentation processes, simultaneous saccharification and fermentation (SSF) has demonstrated the best performance of d-lactic acid production. Because it prevents fructose inhibition and promotes the complete hydrolysis of inulin, the highest d-lactic acid concentration (123.6 ± 0.9 g/L) with a yield of 97.9 % was obtained from 120 g/L inulin by SSF. Moreover, SSF by L. bulgaricus CGMCC 1.6970 offered another distinct advantage with respect to the higher optical purity of d-lactic acid (>99.9 %) and reduced number of residual sugars. The excellent performance of d-lactic acid production from inulin by SSF represents a high-yield method for d-lactic acid production from non-food grains.

To investigate the xylose operon and properties of xylose isomerase and xylulokinase in Bacillus coagulans that can effectively ferment xylose to lactic acid.The xylose operon is widely present in B. coagulans. It is composed of four putative ORFs. Novel xylA and xylB from B. coagulans NL01 were cloned and expressed in Escherichia coli. Sequence of xylose isomerase was more conserved than that of xylulokinase. Both the enzymes exhibited maximum activities at pH 7–8 but with a high temperature maximum of 80–85 °C, divalent metal ion was prerequisite for their activation. Xylose isomerase and xylulokinase were most effectively activated by Ni2+ and Co2+, respectively.Genomic analysis of xylose operon has contributed to understanding xylose metabolism in B. coagulans and the novel xylose isomerase and xylulokinase might provide new alternatives for metabolic engineering of other strains to improve their fermentation performance on xylose.

Phenylalanine ammonia-lyase (PAL) is one of the most extensively studied enzymes with its crucial role in secondary phenylpropanoid metabolism of plants. Recently, its demand has been increased for aromatic chemical production, but its applications in trans-cinnamic acid production were not much explored. In the present study, a putative PAL gene from Zea mays designated as ZmPAL2 was expressed and characterized in Escherichia coli BL21 (DE3). The recombinant ZmPAL2 exhibited a high PAL activity (7.14 U/mg) and a weak tyrosine ammonia-lyase activity. The optimal temperature of ZmPAL2 was 55 °C, and the thermal stability results showed that about 50 % of enzyme activity remained after a treatment at 60 °C for 6 h. The recombinant ZmPAL2 is a good candidate for the production of trans-cinnamic acid. The vitro conversion indicated that the recombinant ZmPAL2 could effectively catalyze the l-phenylalanine to trans-cinnamic acid, and the trans-cinnamic acid concentration can reach up to 5 g/l.

Corn stover is a potential feedstock for cellulosic ethanol production. In this study, the performances of four chemical pretreatment methods, low-temperature moderate acid (LTMA), high-temperature dilute acid (HTDA), alkali pretreatment (AP), and sulfite pretreatment (SP), were compared in pretreating non-wood bioresource (corn stover) for an efficient saccharification. Under the investigated conditions, LTMA and HTDA could directly convert most xylan into xylose but had lower enzymatic digestibility from forming more fermentation inhibitors. AP pretreatment achieved better delignification, while its effect on removing xylan was very weak. SP pretreatment not only dissolved about 83.75 and 63.14% of xylan and lignin, respectively, but also caused less known inhibitors than acidic pretreatments. More interestingly, a large amount of xylo-oligosaccharides (about 17.12 g/L) was found in spent liquor from SP pretreatment, which is a potential high-value co-product. After 48 h of enzymatic hydrolysis, the cellulose saccharification yields were 24% for the LTMA substrate, 47% for the HTDA substrate, 60% for the SP substrate, and 65% for the AP substrate, respectively. These results suggested that SP pretreatment was a more suitable pretreatment method for bioethanol production of corn stover.

The addition of non-ionic surfactants has recently been confirmed to positively affect the enzymatic hydrolysis of cellulosic materials. However, the functional mechanisms of these surfactants remain unclear. This work investigated the influence of poly(ethylene glycol) (PEG) on the enzymatic hydrolysis of three cellulosic materials, namely, acid steam-exploded corn straw, pure microcrystalline cellulose (Avicel PH101), and bagasse sulfite pulp (BSP). The results showed that PEG addition led to varied effects on the enzymatic hydrolysis of different cellulosic materials. Addition of PEG was most effective on the enzymatic hydrolysis of PH101 and weakly effective on the hydrolysis of BSP. We further investigated PEG concentrations and enzymatic activities in the supernatant during hydrolysis and found that the positive effects of PEG treatment might contribute to its influence on enzyme desorption from different substrates. We also found that the efficiency of PEG depended on its capacity to bind to different substrates. PEG exhibited stronger affinity to pure cellulose than to the two other lignocellulosic substrates. These findings are helpful in further revealing the mechanism of surfactants and improving the enzymatic hydrolysis process.

The purpose of this study was to produce a Trichoderma reesei xylanase (XYN2) in Pichia pastoris and to test its potential application for pulp bleaching. The recombinant xylanase was purified by a two-step process of ultrafiltration and gel filtration chromatography. The molecular mass of the recombinant enzyme was 21 and 25 kDa by SDS–PAGE analysis, due to different glycosylation of the native protein. The optimum pH and temperature of the recombinant XYN2 was 5.0 and 50 °C. Enzyme activity was stable at 50 °C and at pH 5.0–7.0. The bleaching ability of the recombinant xylanase was also studied at 50 °C and pH 6.0, using wheat straw pulp. Biobleaching of the xylanase produced chlorine dioxide savings of up to 60%, while retaining brightness at the control level and led to a lower kappa number and small enhancements in tensile, burst and tear strength of pulp fibers.

Ethylene production from petroleum or natural gas is an energy intensive process. Bio-ethanol catalytic dehydration to ethylene is an attractive alternative for oil based ethylene. Catalytic dehydration conversion of bio-ethanol to ethylene using HZSM-5 modified by 3 wt% rare earth metal (lanthanum) was carried out in a laboratory bioreactor. The physicochemical properties of the catalyst were characterized. The stability test showed that ethanol conversion and selectivity over this catalyst could be maintained above 98% for more than 950 h. The regenerated catalyst also displayed high reactivity and stability of up to 830 h can be obtained. The effects of temperature, liquid hourly space velocity, particle size of catalyst, and bio-ethanol partial pressure on products formation rate were investigated. The external and internal diffusion resistances were eliminated and the kinetic control range was identified. An apparent kinetics model was used to describe the dehydration reaction of ethanol over 3 wt% La-HZSM-5 catalyst, and the kinetic parameters were determined.

•The l-nLDH of B. coagulans NL01 was a thermostable and efficient biocatalyst.•The l-nLDH could catalyze a reversible reaction.•Ca2+, Ba2+, Mg2+ and Mn2+ had the positive effect on the l-nLDH.•The l-nLDH showed higher specific activities toward pyruvate esters.Bacillus coagulans is a homofermentative, acid-tolerant and thermophilic sporogenic lactic acid bacterium, which is capable of producing high yields of optically pure lactic acid. The l-(+)-lactate dehydrogenase (l-LDH) from B. coagulans is considered as an ideal biocatalyst for industrial production. In this study, the gene ldhL encoding a thermostable l-LDH was amplified from B. coagulans NL01 genomic DNA and successfully expressed in Escherichia coli BL21 (DE3). The recombinant enzyme was partially purified and its enzymatic properties were characterized. Sequence analysis demonstrated that the l-LDH was a fructose 1,6-diphosphate-activated NAD-dependent lactate dehydrogenase (l-nLDH). Its molecular weight was approximately 34–36 kDa. The Km and Vmax values of the purified l-nLDH for pyruvate were 1.91 ± 0.28 mM and 2613.57 ± 6.43 μmol (min mg)−1, respectively. The biochemical properties of l-nLDH showed that the specific activity were up to 2323.29 U/mg with optimum temperature of 55 °C and pH of 6.5 in the pyruvate reduction and 351.01 U/mg with temperature of 55 °C and pH of 11.5 in the lactate oxidation. The enzyme also showed some activity in the absence of FDP, with a pH optimum of 4.0. Compared to other lactic acid bacterial l-nLDHs, the enzyme was found to be relatively stable at 50 °C. Ca2+, Ba2+, Mg2+ and Mn2+ ions had activated effects on the enzyme activity, and the enzyme was greatly inhibited by Ni2+ ion. Besides these, l-nLDH showed the higher specificity towards pyruvate esters, such as methyl pyruvate and ethyl pyruvate.

•SSF demonstrated the remarkable advantage over SHF from BSP by strain CC17.•SSF by strain CC17 could lower about 33.3% fungal cellulase dosage over SHF.•Strain CC17 could convert cellobiose to lactic acid without exogenous β-glucosidase.•110 g/L l-lactic acid was obtained in the fed-batch SSF of BSP by strain CC17.The main barriers to cost-effective lactic acid production from lignocellulose are the high cost of enzymes and the ineffective utilization of the xylose within the hydrolysate. In the present study, the thermophilic Bacillus coagulans strain CC17 was used for the simultaneous saccharification and fermentation (SSF) of bagasse sulfite pulp (BSP) to produce l-lactic acid. Unexpectedly, SSF by CC17 required approximately 33.33% less fungal cellulase than did separate hydrolysis and fermentation (SHF). More interestingly, CC17 can co-ferment cellobiose and xylose without any exogenous β-glucosidase in SSF. Moreover, adding xylanase could increase the concentration of lactic acid produced via SSF. Up to 110 g/L of l-lactic acid was obtained using fed-batch SSF, resulting in a lactic acid yield of 0.72 g/g cellulose. These results suggest that SSF using CC17 has a remarkable advantage over SHF and that a potentially low-cost and highly-efficient fermentation process can be established using this protocol.

l-Phenyllactic acid (l-PLA) is a novel antiseptic agent with broad and effective antimicrobial activity. In addition, l-PLA has been used for synthesis of poly(phenyllactic acid)s, which exhibits better mechanical properties than poly(lactic acid)s. However, the concentration and optical purity of l-PLA produced by native microbes was rather low. An NAD-dependent l-lactate dehydrogenase (l-nLDH) from Bacillus coagulans NL01 was confirmed to have a good ability to produce l-PLA from phenylpyruvic acid (PPA). In the present study, l-nLDH gene and formate dehydrogenase gene were heterologously coexpressed in Escherichia coli. Through two coupled reactions, 79.6 mM l-PLA was produced from 82.8 mM PPA in 40 min and the enantiomeric excess value of l-PLA was high (>99%). Therefore, this process suggested a promising alternative for the production of chiral l-PLA.