Production of inulo-oligosaccharides from inulin by recombinant E. coli containing endoinulinase activity

To utilize intracellular endoinulinase for inulo-oligosaccharide (IOS) production from inulin, the endoinulinase gene (inu1) of Pseudomonas sp. was successfully cloned into the plasmid pBR322 by using EcoRI restriction endoinulinase and E. coli HB101 as a host strain. The endoinulinase from E. coli HB101/pKMG50 was constitutively expressed, showing similar reaction modes as compared to those of the original strain. However, some critical differences existed in optimal reaction conditions and oligosaccharide compositions between the two products catalyzed by the native enzyme of original strain and those by intact cells from recombinant cells. The IOS compositions produced by recombinant E. coli were quite different due to the diffusional restriction of the substrate and products within the cell wall. Optimal reaction conditions for batchwise production of IOS were as follow : optimum temperature, 55v°C; pH, 7.5; substrate concentration, 100 g/l inulin; enzyme dosage, 20 units/g substrate. Continuous production of IOS from inulin was also carried out at 50v°C using a bioreactor packed with the recombinant cells immobilized on calcium alginate gel. The optimal feed concentration and the feed flow rate were 100 g/l inulin and 0.6 h^у as a superficial space velocity, respectively. Under the optimum operation conditions, continuous production of IOS was successfully performed with productivity of 166.7 g/l·h for 15 days at 50v°C without significant loss of initial activity.     

Optimization of enzymatic hydrolysis of mango kernel starch by response surface methodology

Amyloglucosidase (EC 3.2.1.3) from Aspergillus niger was employed for the saccharification of mango (Mangifera indica Linn) kernel starch. Response surface methodology based on a three-level three-factor Box-Behnken design was employed to optimize the important process variables such as substrate concentration (137.5-412.5 mg), enzyme concentration (4-12 mg) and temperature (35-55 °C). The sugar yield increased with both enzyme concentration and temperature, and decreased with substrate concentration. The response surface model indicated optimum conditions (substrate, 137.5 mg; enzyme, 12 mg; temperature, 55 °C) for obtaining 0.4851 mg sugar/mg substrate, which was also verified experimentally.     

Production and purification of α-amylase from hydrogen producing Enterobacter cloacae IIT-BT 08

Enterobacter cloacae IIT-BT08 was found to produce both !-amylase and hydrogen in a batch system using soluble starch as substrate. Incubation time, temperature, pH and substrate concentration for the maximum !-amylase activity (130 U/ml) were 8 h, 37 °C, 6.00 and 10 g/l of soluble potato starch respectively. However, the optimum temperature and pH for the crude !-amylase activity were 60 °C and 4 respectively. The maximum rate of hydrogen production was observed at 10th h of fermentation and corresponding hydrogen yield was 7.6 mmol H₂/g soluble potato starch.     

The potential of enzyme recycling during the hydrolysis of a mixed softwood feedstock

Despite recent improvement in cellulase enzymes properties, the high cost associated with the hydrolysis step remains a major impediment to the commercialization of full-scale lignocellulose-to-ethanol bioconversion process. As part of a research effort to develop a commercial process for bioconversion of softwood residues, we have examined the potential for recycling enzymes during the hydrolysis of mixed softwood substrate pretreated by organosolv process. We have used response surface methodology to determine the optimal temperature, pH, ionic strength, and surfactant (Tween 80) concentration for maximizing the recovery of bound protein and enzyme activity from the residual substrates after hydrolysis. Data analysis showed that the temperature, pH and surfactant concentration were the major factors governing enzyme desorption from residual substrate. The optimized conditions were temperature 44.4 °C, pH 5.3 and 0.5% Tween 80. The optimal conditions significantly increased the hydrolysis yield by 25% after three rounds of hydrolysis. This bound enzyme desorption combining with free enzyme re-adsorption is a potential method to recover cellulase enzymes and reduce the cost of enzymatic hydrolysis.     

Enhanced expression of endoinulinase from Aspergillus niger by codon optimization in Pichia pastoris and its application in inulooligosaccharide production

In the present study, the endoinulinase gene (EnInu) from Aspergillus niger CICIM F0620 was optimized according to the codon usage of Pichia pastoris and both the native and the optimized gene were expressed in P. pastoris. Use of the optimized gene resulted in the secretion of recombinant endoinulinase activity that reached 1,349 U ml^?1, 4.18 times that observed using the native gene. This is the highest endoinulinase activity reported to date. The recombinant enzyme was optimally active at pH 6.0 and 60 °C. Moreover, inulooligosaccharides production from inulin was studied using the recombinant enzyme produced from the optimized gene. After 8 h under optimal conditions, which included 400 g l^?1 inulin, an enzyme concentration of 40 U g^?1 substrate, 50 °C and pH 6.0, the inulooligosaccharide yield was 91 %. The high substrate concentration and short reaction time described here should reduce production costs distinctly, compared with the conditions used in previous studies. Thus, this study may provide the basis for the industrial use of this recombinant endoinulinase for the production of inulooligosaccharides.     

Chlorogenate hydrolase-catalyzed synthesis of hydroxycinnamic acid ester derivatives by transesterification, substitution of bromine, and condensation reactions

A chlorogenate hydrolase (EC 3.1.1.42) synthesized 2-phenylethyl caffeate (2-CAPE) from 5-chlorogenic acid (5-CQA) and 2-phenylethyl alcohol (2-PA) (by transesterification), from 5-CQA and 2-phenylethyl bromide (2-PBr) (by substitution of bromine), and from caffeic acid (CA) and 2-PA or 2-PBr (by condensation) as well as hydrolysis of 5-CQA. Some reaction conditions including pH, temperature, substrate and solvent concentrates, and reaction time were optimized for the production of 2-CAPE. A maximal molar yield of 50% was achieved by transesterification, 4.7% by substitution of bromine, and 13% by condensation. Among the parameters studied for optimization, the pH of the buffer solution and concentration of 2-PA or 2-PBr affected the production of 2-CAPE. The optimum pH for the hydrolysis reaction was within the neutral range (pH 6.5), whereas the residual three reactions were only catalyzed within the acidic range (pH 3.0–4.0). The optimum concentrations of 2-PA and 2-PBr for three reactions were 5–70 vol% and no 2-CAPE was produced in the 2-PA or 2-PBr solutions containing powdered enzyme. The enzyme may bind to the caffeoyl moiety of 5-CQA or CA to form an enzyme–substrate complex. It then catalyzes four different reactions corresponding to the reaction conditions.     

Production of α-galactosidase by a novel actinomycete <Emphasis Type="Italic">Streptomyces griseoloalbus</Emphasis> and its application in soymilk hydrolysis

α-Galactosidase production by a newly isolated actinomycete Streptomyces griseoloalbus under submerged fermentation was investigated. The influence of initial pH of medium, incubation temperature, inoculum age and inoculum size on α-galactosidase formation was studied. Various carbon sources were supplemented in the medium to study their effect on enzyme production. The influence of the concentration of locust bean gum on enzyme production also was optimized. Optimization of process parameters resulted in a highest α-galactosidase activity of 20.4 U/ml. The highest α-galactosidase activity was obtained when the fermentation medium with initial pH 6.0 and containing 1% locust bean gum as growth substrate was inoculated with 10% (v/v) of 72 h grown inoculum and incubated at 30°C. The hydrolysis of flatulence-causing oligosaccharides in soymilk by the enzyme was also investigated. Thin layer chromatographic analysis of enzyme-treated soymilk samples showed the complete hydrolysis of soy oligosaccharides liberating galactose, the final product.     

Statistical optimization of lipase catalyzed hydrolysis of methyloleate by response surface methodology

Hydrolysis of methyloleate was optimized using lipase from Chromobacterium viscosum immobilized on IRC-50. The optimization was studied by a statistical methodology using response surface methodology (RSM). The cumulative interactive effect of substrate, water concentration and time was studied in optimizing the hydrolysis of methyloleate. The interactive effect of substrate-time was found to be significant compared to substrate-water and time-water interactions. A well correlation was observed between the optimum values obtained from the response surface contour plots and from the quadratic regression model equation. The optimal values obtained for substrate, water and time were found to be in the experimental range chosen.     

Optimization of enzymatic hydrolysis and fermentation conditions for improved bioethanol production from potato peel residues

The aim of this work was the optimization of the enzyme hydrolysis of potato peel residues (PPR) for bioethanol production. The process included a pretreatment step followed by an enzyme hydrolysis using crude enzyme system composed of cellulase, amylase and hemicellulase, produced by a mixed culture of Aspergillus niger and Trichoderma reesei. Hydrothermal, alkali and acid pretreatments were considered with regards to the enhancement of enzyme hydrolysis of potato peel residues. The obtained results showed that hydrothermal pretreatment lead to a higher enzyme hydrolysis yield compared to both acid and alkali pretreatments. Enzyme hydrolysis was also optimized for parameters such as temperature, pH, substrate loading and surfactant loading using a response surface methodology. Under optimized conditions, 77 g L^?1 of reducing sugars were obtained. Yeast fermentation of the released reducing sugars led to an ethanol titer of 30 g L^?1 after supplementation of the culture medium with ammonium sulfate. Moreover, a comparative study between acid and enzyme hydrolysis of potato peel residues was investigated. Results showed that enzyme hydrolysis offers higher yield of bioethanol production than acid hydrolysis. These results highlight the potential of second generation bioethanol production from potato peel residues treated with onsite produced hydrolytic enzymes. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:397–406, 2017     

Optimization of enzymatic hydrolysis of visceral waste proteins of Catla (Catla catla) for preparing protein hydrolysate using a commercial protease

Protein hydrolysate was prepared from visceral waste proteins of Catla (Catla catla), an Indian freshwater major carp. Hydrolysis conditions (viz., time, temperature, pH and enzyme to substrate level) for preparing protein hydrolysates from the fish visceral waste proteins were optimized by response surface methodology (RSM) using a factorial design. Model equation was proposed with regard to the effect of time, temperature, pH and enzyme to substrate level. An enzyme to substrate level of 1.5% (v/w), pH 8.5, temperature of 50 degrees C and a hydrolysis time of 135 min were found to be the optimum conditions to obtain a higher degree of hydrolysis close to 50% using alcalase. The amino acid composition of the protein hydrolysate prepared using the optimized conditions revealed that the protein hydrolysate was similar to FAO/WHO reference protein. The chemical scores computed indicated methionine to be the most limiting amino acid. The protein hydrolysate can well be used to meet the amino acid requirements of juvenile common carp and hence has the potential for application as an ingredient in balanced fish diets.     

Culture conditions for the production of esterase from Lactobacillus casei CL96

Culture conditions in growth and esterase production by a newly isolated Lactobacillus casei CL96 were investigated using a dextrose-free MRS medium supplemented with different sugars in a 2 l fermentor at different pHs (4.0-9.0) and temperatures (20-50°C). The optimal growth was obtained in basal MRS medium containing 1% (w/v) lactose at pH 7.0 and 30°C. The maximal esterase production was obtained intracellularly during the late logarithmic phase, but during the stationary phase, the esterase activity was released in the culture medium. The enzyme activity was maximal at pH 7.0 and 37°C. Among various substrates (C₂-C₁₆) tested, the highest activity was towards C₆ and C₈. Though the enzyme was produced constitutively, the tributylin induced the enzyme production by 2.5 fold. L. casei CL96 esterase was very active at neutral pH and ambient temperature and might be suitable for biotechnological applications in the dairy industry.     

Immobilization of cellulase onto electrospun polyacrylonitrile (PAN) nanofibrous membranes and its application to the reducing sugar production from microalgae

This study demonstrates a method to prepare an immobilized cellulase by using an electrospun polyacrylonitrile (PAN) nanofibrous membrane as the support. To obtain an immobilized cellulase with high hydrolytic activity, the immobilization conditions including activation time, enzyme concentration, immobilization time, and temperature were optimized. Under those conditions, the immobilized cellulase possessed a protein loading of 30 mg/g-support and a specific activity of 3.2 U/mg-protein. After immobilization, the enzymatic stability of cellulase against pH and thermal stresses was improved. Fourier transform infrared spectroscopy (FTIR) measurements also revealed that the cellulase was covalently bonded to the supports. The immobilized cellulase was then used to hydrolyze cell wall of microalgae for the production of reducing sugars. Analyses using response surface methodology (RSM) show that the hydrolysis yield was affected by the reaction temperature, pH, and substrate/cellulase mass ratio, and a hydrolysis yield of 60.86% could be obtained at 47.85 °C, pH 5.82, and a substrate/cellulase mass ratio of 40 g-substrate/g-cellulase. This result suggests that the proposed scheme for the cellulase immobilization has great potential for the application to the reducing sugar production.     

牦牛骨蛋白的酶解条件研究

以蛋白质水解度为评价指标,辅以固形物溶出率,比较了中性蛋白酶、菠萝蛋白酶和木瓜蛋白酶对牦牛骨蛋白的水解效果,研究了酶用量、料液比(底物浓度)、酶解时间对水解度的影响,采用正交试验对酶解条件进行了优化。结果显示,木瓜蛋白酶是牦牛骨蛋白水解的适宜催化剂。在一定条件下,样品水解度随酶用量和酶解时间的增加而增大,底物浓度过低或过高均不利于原料中蛋白质的酶解。木瓜蛋白酶水解牦牛骨蛋白最佳条件为:酶解温度60℃,酶解时间8 h,酶用量3500 U/g蛋白质,料液比1:25(g:m l)。     

Verticillium lecanii spore production in solid-state and liquid-state fermentations

Verticillium lecanii has been recognized as an entomopathogen with high potential in biological control of pests. Two types of cultivation methods, the solid-state fermentation (SSF) and the liquid-state fermentation (LSF), were examined for V. lecanii. In SSF, the substrate types including rice, rice bran, rice husk, and the mixtures of these components were tested. The results showed that both cooked rice with appropriate water addition and rice bran gave significantly higher spore production of 1.5 2 10⁹ spores/g substrate and 1.4 2 10⁹ spores/g substrate, respectively. In LSF, SMAY liquid medium was used as a base, and the effects of environmental conditions on the spore production of V. lecanii were investigated. From the time course study, on the 9th day the spore yield reached 1.2 2 10⁹ spores/ml of broth at 24v°C, 150 rpm for this strain. A series of medium volumes in the shaker-flask have been tested for the requirement of aeration. The largest surface aeration test, one tenth of the medium volume in the shaker-flask for cultivation, gave the highest spore count. The optimal pH value was tested and the initial pH 5 in the SMAY medium produced a high spore density. Finally, V. lecanii spores from SSF and LSF were different in size, shape, and size distribution; while mean spore length from SSF was 6.1 7m, and mean spore length from LSF was 5.0 7m.     

影响纳豆激酶酶促反应速度因素的研究

利用分光光度法对由纳豆菌发酵产生的纳豆激酶 (NK)进行了动力学性质的研究。以双倒数作图法 (L -B作图法 )求取Km。采用单因素试验法和正交试验法研究了底物浓度、酶浓度、温度、pH值对酶促反应速度的影响。结果表明该纳豆激酶的Km值为 3.4 98× 1 0 -6g·mL-1 ,当水解时间为 1 0min时 ,最适底物浓度为 1 6mg·mL-1 ,最适温度为 6 0℃ ,最适 pH为 8.0。     

Pretreatment of lignocellulosic biomass using ultrasound aiming at obtaining fermentable sugar

This study aims to evaluate the activity of the cellulase enzyme forward the use of ultrasound technology in different conditions of temperature, pH and exposure time, as well, to match the steps of pretreatment and enzymatic hydrolysis in one step. A central composite design (CCRD) and response surface analysis were used to evaluate the effect of ultrasound power, temperature and pH on enzyme activity. Optimum condition in the studied range was 30% for ultrasound power, pH 4.6 and 50?°C, yielding an enzyme activity of 15.5 UPF/mL. From this, we carried out kinetics of enzymatic hydrolysis on filter paper and bagasse malt, in optimized conditions. Total reducing sugars (TRS) were 3.85 and 0.46?mg/mL when the filter paper and bagasse malt were used as substrate, respectively. Ultrasound showed to be a good technology to increase the enzyme activity aiming to intensify enzymatic processes.     

Chitinase production by a newly isolated thermotolerant Paenibacillus sp. BISR-047

Chitinase is one of the important mycolytic enzymes with industrial significance, and is produced by a number of organisms, including bacteria. In this study, we describe isolation, characterization and media optimization for chitinase production from a newly isolated thermotolerant bacterial strain, BISR-047, isolated from desert soil and later identified as Paenibacillus sp. The production of extracellularly secreted chitinase by this strain was optimized by varying pH, temperature, incubation period, substrate concentrations, carbon and nitrogen source,etc. The maximum chitinase production was achieved at 45 °C with media containing (in g/l) chitin 2.0, yeast extract 1.5, glycerol 1.0, and ammonium sulphate 0.2 % (media pH 7.0). A three-fold increase in the chitinase production (712 IU/ml) was found at the optimized media conditions at 6 days of incubation. The enzyme showed activity at broad pH (3–10) and temperature (35–100 °C) ranges, with optimal activity displayed at pH 5.0 and 55 °C, respectively. The produced enzyme was found to be highly thermostable at higher temperatures, with a half-life of 4 h at 100 °C.     

Joint aerobic biodegradation of wastewater from table olive manufacturing industries and urban wastewater

Wastewater generated in the elaboration of table olives has been treated using activated sludge from a municipal wastewater plant after adequate acclimation. To avoid bactericide properties of some chemical structures present in this type of effluents, synthetic urban wastewater has been used to dilute the original wastewater. The main parameters affecting efficiency of biological processes have been studied. Thus, initial biomass concentration, temperature up to 303 K (upper working temperature limit = 313 K) and initial substrate concentration exerted a positive influence on COD degradation rate. The optimum pH was found to be around 7, experiencing a slight inhibition on cell activity at pH 4. Under the experimental conditions investigated other parameters like polyphenol content, absorbance at 254 nm and total organic carbon were also reduced to some extent. Only nitrates amount was increased after the biological process took place. A kinetic model based on Monod equation was proposed and applied to experimental results. The maximum specific growth rate was calculated by means of the aforementioned kinetic model. The value of this parameter as a function of temperature was fitted to an Arrhenius expression, w_max = 9.43 2 10¹⁰ exp(72021/RT) h^у (R in J mol^у K^у283 K < T < 303 K, pH , 7-10).     

Process Modeling of Enzymatic Hydrolysis of Wet-Exploded Corn Stover

The aim of this work was to investigate the optimal process conditions leading to high glucose yield (over 80 %) after wet explosion (WEx) pretreatment and enzymatic hydrolysis. The study focused on determining the “sweet spot” where the glucose yield obtained is optimized compared to the cost of the enzymes. WEx pretreatment was conducted at different temperatures, times, and oxygen concentrations to determine the best WEx pretreatment conditions for the most efficient enzymatic hydrolysis. Enzymatic hydrolysis was further optimized at the optimal conditions using central composite design of response surface methodology with respect to two variables: Cellic® CTec2 loading [5 to 40 mg enzyme protein (EP)/g glucan] and substrate concentration (SC) (5 to 20 %) at 50 °C for 72 h. The most efficient and economic conditions for corn stover conversion to glucose were obtained when wet-exploded at 170 °C for 20 min with 5.5 bar oxygen followed by enzymatic hydrolysis at 20 % SC and 15 mg EP/g glucan (5 filter paper units) resulting in a glucose yield of 84 %.     

Protein hydrolysate from visceral waste proteins of Catla (Catla catla): optimization of hydrolysis conditions for a commercial neutral protease

Protein hydrolysate was prepared from visceral waste proteins of an Indian freshwater major carp, Catla catla. Hydrolysis conditions (viz., time, temperature and enzyme to substrate level) for preparing protein hydrolysates from the fish visceral waste proteins using in situ pH of the visceral mass were optimized by response surface methodology (RSM) by employing a factorial design. The regression coefficient close to 1.0, observed during both experimental and validation runs, indicated the validity of prediction model. An enzyme to substrate level of 1.25 % (v/w), temperature of 55 degrees C and a hydrolysis time of 165 min were found to be the optimum conditions to obtain a higher degree of hydrolysis of >48% using multifect-neutral. The amino acid composition of the protein hydrolysate prepared using the optimized conditions revealed that the protein hydrolysate was similar to FAO/WHO reference protein. The chemical scores computed indicated methionine to be the most limiting amino acid. The protein hydrolysate has the potential for application as an ingredient in balanced fish diets.