Pretreatment of starch raw materials and their enzymatic hydrolysis by immobilized glucoamylase

The pretreatment of starch raw materials such as sweet potato, potato and cassava has been carried out using various types of crusher, viz juice mixer, homogenizer and high-speed planetary mill. The effect of pretreatment of the materials on their enzymatic hydrolysis was studied. High-speed planetary mill treatment was the most effective and comparable with heat treatment (pasting). Various crushing times were used to examine the effect of crushing by mill treatment on the enzymatic hydrolysis. In the enzymatic hydrolysis of cassava, the use of both cellulase [1,4-(1,3; 1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] and glucoamylase [1,4-α-d-glucan glucohydrolase, EC 3.2.1.3] enhanced the d-glucose yield. The immobilization of glucoamylase was studied by radiation polymerization of hydrophilic monomers at low temperature, and it was found that enzymatic activity of the immobilized glucoamylase particles varied with monomer concentration and particle size. Starchy raw materials pretreated with the mill can be efficiently hydrolysed by immobilized glucoamylase.     

Immobilization of glucoamylase on DEAE-cellulose activated with chloride compounds

Partially purified glucoamylase (1,4-α-d-glucan glucohydrolase, EC 3.2.1.3) from Aspergillus niger NRRL 330 has been immobilized on DEAE-cellulose activated with cyanuric chloride in 0.2 m acetate buffer, pH 4.2. In the matrix-bound glucoamylase, enzyme yield was 20 mg g^?1 of support, corresponding to 40 200 units g^?1 of DEAE support. Binding of the enzyme narrows the pH optimum from 3.8–5.2 to 3.6. Thermal stability of the bound glucoamylase enzyme was decreased although it showed a higher temperature optimum (70°C) than the free form (55°C). The rate of reaction of glucoamylase was also changed after immobilization. V_max values for free and bound enzyme were 36.6 and 22.6 μmol d-glucose ml^?1 min^?1 and corresponding K_m values were 3.73 and 4.8 g l^?1 respectively. Free and immobilized enzyme when used in the saccharification process gave 84 and 56% conversion of starch to d-glucose, respectively. The bound enzyme was quite stable and in the batch process it was able to operate for about five cycles without any loss of activity.     

Characterization of an organic solvent‐tolerant thermostable glucoamylase from a halophilic isolate,Halolactibacillus sp. SK71 and its application in raw starch hydrolysis for bioethanol production

A halophilic bacterium Halolactibacillus sp. SK71 producing extracellular glucoamylase was isolated from saline soil of Yuncheng Salt Lake, China. Enzyme production was strongly influenced by the salinity of growth medium with maximum in the presence of 5% NaCl. The glucoamylase was purified to homogeneity with a molecular mass of 78.5 kDa. It showed broad substrate specificity and raw starch hydrolyzing activity. Analysis of hydrolysis products from soluble starch by thin‐layer chromatography revealed that glucose was the sole end‐product, indicating the enzyme was a true glucoamylase. Optimal enzyme activity was found to be at 70°C, pH 8.0, and 7.5% NaCl. In addition, it was highly active and stable over broad ranges of temperature (0–100°C), pH (7.0–12.0), and NaCl concentration (0–20%), showing excellent thermostable, alkali stable, and halotolerant properties. Furthermore, it displayed high stability in the presence of hydrophobic organic solvents. The purified glucoamylase was applied for raw corn starch hydrolysis and subsequent bioethanol production using Saccharomyces cerevisiae. The yield in terms of grams of ethanol produced per gram of sugar consumed was 0.365 g/g, with 71.6% of theoretical yield from raw corn starch. This study demonstrated the feasibility of using enzymes from halophiles for further application in bioenergy production. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1262–1268, 2014     

Characterization of glucoamylase adsorption to raw starch

The adsorption of Aspergillus niger glucoamylase forms (GA-I and GA-II) to raw corn starch was studied as a function of pH, ionic strength, and temperature. A three-parameter model was developed to account for the specific and nonspecific adsorption of GA-I to starch. The adsorption of the GA-II form to raw starch was weak and independent of the pH and ionic strength of the mixture. GA-I was bound strongly to the starch surface, with association constant values ranging from 2 to 5 × 10⁶ M⁻¹. Maximum adsorption capacities (saturation concentrations) Q_max for GA-I were affected by pH, inonic strength, and temperature and varied between 1.6 and 4.3 mg protein g⁻¹ starch. The tightly bound GA-I could be specifically eluted from the starch surface with maltose, maltodextrin, or soluble starch. The adsorption of GA-II to starch in the presence of acarbose (glucoamylase activity inhibitor) indicated that the active site participates minimally in the adsorption process. The comparison of the distribution coefficients of GA-I and GA-II showed that the starch-binding domain, present only in GA-I, increases the affinity of GA-I for starch by two orders of magnitude.     

Growth and amylolytic activity of Aureobasidium pullulans in starch-limited culture

One strain of the yeast-like fungus Aureobasidium pullulans has been found which converts starch into biomass with a high yield (Y_starch = 0.590) and releases glucoamylase (1,4-α-d-glucan glucohydrolase EC 3.2.1.3) to a certain extent (ca. 2.2–2.3 U ml^?1) into the culture medium. The rate of starch hydrolysis seems to be high enough so as not to limit the specific growth rate.     

Multiple forms of a glucoamylase inhibitory factor fromAspergillus niger and its elution after adsorption onto raw wheat starch

An inhibitory factor (IF) fromAspergillus niger, that inhibited the action of glucoamylase on raw starch, was adsorbed tightly onto raw starch but was almost completely desorbed by 0.02m sodium borate. The IF was a glycoprotein and was partially purified by ion exchange chromatography into three active fractions.     

Immobilization of glucoamylase on porous silicas

The influence of the pore structure of silica carriers (macroporous silica gels, silochromes and porous glasses) on the catalytic activity of immobilized glucoamylase (exo 1,4-α-d-glucosidase, 1,4-α-d-glucan glucohydrolase EC 3.2.1.3) has been studied. The dependence of the immobilized glucoamylase activity, in units g^?1, on the carrier pore diameter was found to pass through a maximum within a range 70–100 nm. Macroporous silica gels can be used with success as carriers for glucoamylase immobilization instead of porous glasses and silochromes.     

Investigation of the binding mechanism of glucoamylase to alkylamine derivatives of titanium(IV)-activated porous inorganic supports

Glucoamylase (exo-1,4-α-d-glucosidase, EC 3.2.1.3) has been coupled to several porous silica matrices by a new covalent process using alkylamine derivatives of titanium(IV)-activated supports. In order to investigate the interaction of the titanium element with the silanol groups of the inorganic matrices, activation was performed at different times, using titanium(IV) chloride, either pure or as a 15% w/v solution, in 15% w/v hydrochloric acid at 25, 45 and 80°C, followed by washing with sodium acetate buffer (0.02m, pH 4.5) or chloroform. Using pure TiCl₄, the highest activities of all preparations were obtained at 80°C and with acetate buffer washing, resulting from a higher content of titanium coating of the carrier. When activation was performed in aqueous TiCl₄ solution, followed by a drying step, the highest activity was obtained with preparations washed with chloroform, with or without amination. When reacting pure TiCl₄ with controlled pore glass (CPG) and with porous silica (Spherosil), colour formation was observed after reaction of glutaraldehyde with the aminated support. This did not happen when Celite was used as the support. As a criterion for comparison of the different immobilized enzyme preparations, the concept of an ‘instability factor’, which measures the percentage of immobilized enzyme activity due to release of enzyme into solution, is introduced. Instability factors of immobilized enzyme preparations on Celite were always higher than those obtained with the other matrices, confirming that there was no covalent coupling of the enzyme to Celite. However, when the activation was performed with aqueous TiCl₄ solution with drying, Schiff's base formation was observed in all preparations and very stable immobilized enzyme preparations were obtained. The results of the activation of controlled pore glass and porous silica with pure titanium(IV) chloride suggest the existence of a true reaction between the titanium element and the silanol groups of these carriers by formation of a bridge, Si-O-Ti, while with the titanium(IV) chloride solution in hydrochloric acid, a coating of hydrous titanium(IV) oxide is obtained.     

Engineering Saccharomyces cerevisiae for direct conversion of raw,uncooked or granular starch to ethanol

The production of raw starch-degrading amylases by recombinant Saccharomyces cerevisiae provides opportunities for the direct hydrolysis and fermentation of raw starch to ethanol without cooking or exogenous enzyme addition. Such a consolidated bioprocess (CBP) for raw starch fermentation will substantially reduce costs associated with energy usage and commercial granular starch hydrolyzing (GSH) enzymes. The core purpose of this review is to provide comprehensive insight into the physiological impact of recombinant amylase production on the ethanol-producing yeast. Key production parameters, based on outcomes from modifications to the yeast genome and levels of amylase production, were compared to key benchmark data. In turn, these outcomes are of significance from a process point of view to highlight shortcomings in the current state of the art of raw starch fermentation yeast compared to a set of industrial standards. Therefore, this study provides an integrated critical assessment of physiology, genetics and process aspects of recombinant raw starch fermenting yeast in relation to presently used technology. Various approaches to strain development were compared on a common basis of quantitative performance measures, including the extent of hydrolysis, fermentation-hydrolysis yield and productivity. Key findings showed that levels of α-amylase required for raw starch hydrolysis far exceeded enzyme levels for soluble starch hydrolysis, pointing to a pre-requisite for excess α-amylase compared to glucoamylase for efficient raw starch hydrolysis. However, the physiological limitations of amylase production by yeast, requiring high biomass concentrations and long cultivation periods for sufficient enzyme accumulation under anaerobic conditions, remained a substantial challenge. Accordingly, the fermentation performance of the recombinant S. cerevisiae strains reviewed in this study could not match the performance of conventional starch fermentation processes, based either on starch cooking and/or exogenous amylase enzyme addition. As an alternative strategy, the addition of exogenous GSH enzymes during early stages of raw starch fermentation may prove to be a viable approach for industrial application of recombinant S. cerevisiae, with the process still benefitting from amylase production by CBP yeast during later stages of cultivation.     

Direct fermentation of raw starch using a Kluyveromyces marxianus strain that expresses glucoamylase and Alpha‐amylase to produce ethanol

Raw starch and raw cassava tuber powder were directly and efficiently fermented at elevated temperatures to produce ethanol using the thermotolerant yeast Kluyveromyces marxianus that expresses α‐amylase from Aspergillus oryzae as well as α‐amylase and glucoamylase from Debaryomyces occidentalis. Among the constructed K. marxianus strains, YRL 009 had the highest efficiency in direct starch fermentation. Raw starch from corn, potato, cassava, or wheat can be fermented at temperatures higher than 40°C. At the optimal fermentation temperature 42°C, YRL 009 produced 66.52 g/L ethanol from 200 g/L cassava starch, which was the highest production among the selected raw starches. This production increased to 79.75 g/L ethanol with a 78.3% theoretical yield (with all cassava starch were consumed) from raw cassava starch at higher initial cell densities. Fermentation was also carried out at 45 and 48°C. By using 200 g/L raw cassava starch, 137.11 and 87.71 g/L sugar were consumed with 55.36 and 32.16 g/L ethanol produced, respectively. Furthermore, this strain could directly ferment 200 g/L nonsterile raw cassava tuber powder (containing 178.52 g/L cassava starch) without additional nutritional supplements to produce 69.73 g/L ethanol by consuming 166.07 g/L sugar at 42°C. YRL 009, which has consolidated bioprocessing ability, is the best strain for fermenting starches at elevated temperatures that has been reported to date. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:338–347, 2014     

Starch fermentation by recombinant saccharomyces cerevisiae strains expressing the alpha-amylase and glucoamylase genes from lipomyces kononenkoae and saccharomycopsis fibuligera

Lipomyces kononenkoae and Saccharomycopsis fibuligera possess highly efficient alpha-amylase and/or glucoamylase activities that enable both of these yeasts to utilize raw starch as a carbon source. Eight constructs containing the L. kononenkoae alpha-amylase genes (LKA1 and LKA2), and the S. fibuligera alpha-amylase (SFA1) and glucoamylase (SFG1) genes were prepared. The first set of constructs comprised four single gene cassettes each containing one of the individual amylase coding sequences (LKA1, LKA2, SFA1 or SFG1) under the control of the phosphoglycerate kinase gene (PGK1) promoter and terminator, while the second set comprised two single cassettes containing SFA1 and SFG1 linked to their respective native promoters and terminators. The third set of constructs consisted of two double-gene cassettes, one containing LKA1 plus LKA2 under the control of the PGK1 promoter and terminator, and the other SFA1 plus SFG1 controlled by their respective native promoters and terminators. These constructs were transformed into a laboratory strain Saccharomyces cerevisiae (Sigma1278b). Southern-blot analysis confirmed the stable integration of the different gene constructs into the S. cerevisiae genome and plate assays revealed amylolytic activity. The strain expressing LKA1 and LKA2 resulted in the highest levels of alpha-amylase activity in liquid media. This strain was also the most efficient at starch utilization in batch fermentations, utilizing 80% of the available starch and producing 0.61g/100 mL of ethanol after 6 days of fermentation. The strain expressing SFG1 under the control of the PGK1 expression cassette gave the highest levels of glucoamylase activity. It was shown that the co-expression of these heterologous alpha-amylase and glucoamylase genes enhance starch degradation additively in S. cerevisiae. This study has resulted in progress towards laying the foundation for the possible development of efficient starch-degrading S. cerevisiae strains that could eventually be used in consolidated bioprocessing, and in the brewing, whisky, and biofuel industries.     

原生质体融合构建直接利用淀粉产衣康酸菌株

采用衣康酸高产菌株栖土曲霉T-730的原生质体,与葡萄糖淀粉酶产生菌黑曲霉Ni-5k的原生质体进行融合处理.在以30%PEG6000为促融剂,30℃保温20min的融合条件下进行融合,所得异核体经诱导获得3株稳定的融合株(F3,F9,F11).以生淀粉为唯一碳源,对F3,F9,F11的发酵性能测定表明,F3在发酵过程中积累葡萄糖淀粉酶和衣康酸;对F3进行发酵条件初步探讨的结果表明,以10%生淀粉为碳源,连续发酵6d,F3的衣康酸产酸率达40.9mg/mL,对供给淀粉的转化率为40.9%.     

玉米原料无蒸煮酒精发酵工艺的研究

在玉米原料无蒸煮酒精发酵过程中,添加少量的纤维素酶,酸性蛋白酶可提高糖化酶对生淀粉的糖化作用,减少糖化酶用量。在料水比１：２．５,糖化酶加量２００ｕ／ｇ,纤维素酶加量５ｕ／ｇ,酸性蛋白酶加量０．０１％,３０℃,ｐＨ３．５条件下,Ｎｏ．２１４菌株经９６ｈ发酵,醪液酒精度达１２．８％,淀粉利用率达９２．１％。     

Different behavior towards raw starch of three forms of glucoamylase from a Rhizopus sp

Three forms of glucoamylase [EC 3.2.1.3] of a Rhizopus sp., Gluc1 (M.W. 74,000), Gluc2 (M.W. 58,600), and Gluc3 (M.W. 61,400), which have similar pH optima and specific activities towards soluble starch were studied as to their behavior towards raw starch. The pH optima for raw starch digestion were different, that is, 4.5 for Gluc1 and 5.0 for both Gluc2 and Gluc3. All the enzymes digested raw starch almost completely but at quite different rates; Gluc2 and Gluc3, which lack the N-terminal portions of Gluc1, were 22 and 25 times less effective, respectively, for raw starch digestion than Gluc1. Of the three enzymes, only Gluc1 tightly bound to raw starch. Binding of Gluc1 to raw starch occurred pH-dependently with a broad pH optimum of 4.5-5.5, but temperature and ionic strength affected it only slightly and little, respectively. The binding constant of Gluc1 for raw starch at pH 5.0 and 4 degrees C was estimated to be 1.2 X 10(5) M-1. Fragment H (M.W. 16,700), presumably released from the N-terminal part of Gluc1, not only bound to raw starch itself but also inhibited the binding of Gluc1 to raw starch. pap-Gluc (M.W. 57,000) and chymo-Gluc (M.W. 64,000), which are papain- and chymotrypsin-modified Gluc1, respectively, and lack the N-terminal portions of Gluc1, resembled Gluc2 and Gluc3 in raw starch binding as well as digestion. All these results indicate that Gluc1 has a raw starch-binding site, different from the active center, in the N-terminal region. Various substrates and analogs inhibited the binding of Gluc1 to raw starch, presumably due to steric hindrance.     

Aspergillus sp. RSD生淀粉糖化酶的分离纯化及酶学性质

【目的】纯化得到一种生淀粉糖化酶,并对其酶学性质进行分析。【方法】从曲霉RSD发酵液中,经过硫酸铵分级盐析,HiPrep DEAE FF16/10弱阴离子交换层析,凝胶过滤层析,Hiprep 16/10 source 30S阳离子交换层析最终纯化出一种电泳纯的生淀粉酶。【结果】粗酶液纯化倍数为12.65倍,活力回收率为9.02%,SDS-PAGE结果显示该酶的相对分子质量约为82 kD。对该酶的酶学性质分析结果表明,该酶最适作用温度为50°C,在50°C以下稳定性很好,对高温较为敏感;最适作用pH为4.5,在pH 3.5-7.0范围内酶活力较为稳定,在40°C、pH 4.6条件下以可溶性淀粉为底物时的Km值和Vmax值分别为7.44 g/L和1.45 g/(L·min);金属离子对酶活性的影响试验表明,Fe2+对该酶具有显著激活效果,EDTA、Cu2+、K+对该酶酶活力有不同程度的抑制作用;底物特异性研究表明该酶对麦芽糊精具有较高酶活力。【结论】与市售糖化酶及生淀粉糖化酶相比,该酶对生淀粉的降解能力更高,在工业应用上有较好的前景。     

Investigation of the operational stabilities and kinetics of glucoamylase immobilized on alkylamine derivatives of titanium(IV)-activated porous inorganic supports

Glucoamylase (exo-1,4-α-d-glucosidase, EC 3.2.3.1) was coupled to several porous silica matrices by an improved metal-link/chelation process using alkylamine derivatives of titanium(IV)-activated supports. In order to select the titanium activation procedure which gave stable enzyme preparations, long-term stability tests were performed. The immobilized glucoamylase preparations, in which the carrier was activated to dryness with a 15% w/v TiCl₄ solution, displayed very stable behaviour, with half-lives of ～60 days. The optimum operating conditions were determined for these preparations. There are significant differences between the behaviour of the immobilized enzyme and the free enzyme. The apparent K_m increased on immobilization due to diffusional resistances. The pH optimum for the immobilized preparation showed a slight shift to acid pH relative to that of the soluble enzyme. Also, the optimum temperature descreased to 60°C after immobilization. In order to test Michaelis-Menten kinetics at high degrees of conversion, time-course analysis of soluble starch hydrolysis was performed. It was observed that simple Michaelis-Menten kinetics are not applicable to the free/immobilized glucoamylase-starch system at high degrees of conversion.     

Ethanol production from native cassava starch by a mixed culture of Endomycopsis fibuligera and Zymomonas mobilis

The conversion of starch from unhydrolyzed cassava flour to ethanol by a pure culture of Endomycopsis fibuligera and by a co-culture of this amylolytic yeast and the bacterium Zymomonas mobilis was studied. The best overall results were obtained using the mixed culture. After 96 h of fermentation of a medium containing 150 g/l initial cassava starch, an ethanol concentration of 31.4 g/l, a productivity of 0.33 g ethanol/l × h and a yield of 0.21 g ethanol/g initial starch were reached. The highest yield (0.37 g/g) was obtained after 48 h when using a medium containing 50 g/l initial starch.     

Immobilization of glucoamylase from Aspergillus niger on poly(ethylenimine)-coated non-porous glass beads

Glucoamylase (1,4-α-d-glucan glucohydrolase, EC 3.2.1.3) from A. niger was immobilized on cationic nonporous glass beads (13–44 μm) by electrostatic adsorption followed by rosslinking with glutaraldehyde. Over 80% of the enzyme's total soluble activity was expressed upon immobilization. d-Glucose production from maltodextrins was virtually complete, suggesting that the lack of pores can eliminate the problem of product reversion. Immobilized glucoamylase showed decreased stability upon heating, compared with the soluble enzyme.