Starch digestion and adsorption by β-amylase of Emericella nidulans (Aspergillus nidulans)

B. CHATTERJEE, A. GHOSH AND A. DAS. 1992. A mutant strain of Emericella nidulans MNU 82 was isolated by multistep mutation. The β-amylase produced by the mutant was able to digest raw starch. It was readily and strongly adsorbed onto raw starch at pH 5.0. The enzyme to starch ratio was 1950 U/g starch. The enzyme showed no correlation between the capacity of raw starch digestion and adsorption of the enzyme.     

Production and Characteristics of Raw-Starch-Digesting alpha-Amylase from a Protease-Negative Aspergillus ficum Mutant

Mutational experiments were carried out to decrease the protease productivity of Aspergillus ficum IFO 4320 by using N-methyl-N'-nitro-N-nitrosoguanidine. A protease-negative mutant, M-33, exhibited higher alpha-amylaseactivity than the parent strain under submerged culture at 30 degrees C for 24 h. About 70% of the total alpha-amylase activity in the M-33 culture filtrate was adsorbed onto starch granules. The electrophoretically homogeneous preparation of raw-starch-adsorbable alpha-amylase (molecular weight, 88,000), acid stable at pH 2, showed intensive raw-starch-digesting activity, dissolving corn starch granules completely. The preparation also exhibited a high synergistic effect with glucoamylase I. A mutant, M-72, with higher protease activity produced a raw cornstarch-unadsorbable alpha-amylase. The purified enzyme (molecular weight, 54,000), acid unstable, showed no digesting activity on raw corn starch and a lower synergistic effect with glucoamylase I in the hydrolysis of raw corn starch. The fungal alpha-amylase was therefore divided into two types, a novel type of raw-starch-digesting enzyme and a conventional type of raw-starch-nondigesting enzyme.     

Raw Starch-digestive Chitin-immobilized Amylase from a Protease—Glycosidase-less Mutant of Aspergillus awamori var. kawachi

The α-amylase and glucoamylase produced by a protease-, glycosidase-less mutant HF-15 of Aspergillus awamori var. kawachi were found to be adsorbable onto chitin. This adsorption was pH-independent, different from the adsorption onto raw corn starch. The binding between amylases and chitin was so tight that a chitin-immobilized amylase was obtained without the aid of a cross linking agent, glutaraldehyde, and it retained more than 90% of the original activity of the free enzyme. The immobilized amylase digested gelatinized potato starch, glycogen and even raw corn starch to the same high extent as glucose similar to the free enzyme, but it was different from the unbound crude enzyme in the lack of transglucosidase activity, and slightly different in pH- and thermo-stabilities. An experiment using the immobilized amylase for alcohol fermentation demonstrated the possibility of recycling the enzyme for raw starch saccharification.     

Production and Characteristics of Raw-Starch-Digesting α-Amylase from a Protease-Negative Aspergillus ficum Mutant

Mutational experiments were carried out to decrease the protease productivity of Aspergillus ficum IFO 4320 by using N-methyl-N′-nitro-N-nitrosoguanidine. A protease-negative mutant, M-33, exhibited higher α-amylaseactivity than the parent strain under submerged culture at 30°C for 24 h. About 70% of the total α-amylase activity in the M-33 culture filtrate was adsorbed onto starch granules. The electrophoretically homogeneous preparation of raw-starch-adsorbable α-amylase (molecular weight, 88,000), acid stable at pH 2, showed intensive raw-starch-digesting activity, dissolving corn starch granules completely. The preparation also exhibited a high synergistic effect with glucoamylase I. A mutant, M-72, with higher protease activity produced a raw cornstarch-unadsorbable α-amylase. The purified enzyme (molecular weight, 54,000), acid unstable, showed no digesting activity on raw corn starch and a lower synergistic effect with glucoamylase I in the hydrolysis of raw corn starch. The fungal α-amylase was therefore divided into two types, a novel type of raw-starch-digesting enzyme and a conventional type of raw-starch-nondigesting enzyme.     

The effects of reaction conditions on the production of γ-cyclodextrin from tapioca starch by using a novel recombinant engineered CGTase

A novel mutant enzyme namely H43T CGTase can produce up to 39% γ-cyclodextrin (γ-CD) compared to the native enzyme which produces only 10% γ-CD. The effect of the reaction conditions on γ-CD production was studied using this mutant CGTase. The effects of substrate–buffer combination, starch pretreatment and concentration, pH, additives and finally the use of a debranching enzyme improved the γ-CD ratio further. The tapioca–acetate pair gave the highest conversion (16% conversion) among four types of starch and four buffer system combinations. Gelatinized starch was preferred compared to raw tapioca starch in producing a high percentage of γ-CD and conversion rate. Higher pH especially pH 8–9 led to a higher proportion of γ-CD, and was relatively more apparent when the concentration of starch was increased. Forty-six percent γ-CD was produced using 2.5% gelatinized tapioca starch at pH 8. Pullulanase enzyme was found to be useful in reducing the viscosity of tapioca starch paste thus increasing the efficiency of utilization of starch by CGTase by at least 20- to 30-fold. Up to 48% γ-CD can be produced when 4% pullulanase-pretreated tapioca starch was reacted with the CGTase mutant. It was also found that the supplementation of the reaction mixture with glucose, toluene, or cyclododecanone improved the γ-CD yield by 42.2, 46.4, 43.4, and 43.4%, respectively. All the parameters involved have been shown to affect the product specificity of the mutant H43T CGTase transglycosylation mechanism.     

Improved production of an enzyme that hydrolyses raw yam starch by Penicillium sp. S-22 using fed-batch fermentation

A newly isolated strain, Penicillium sp. S-22, was used to produce an enzyme that hydrolyses raw yam starch [raw yam starch digesting enzyme (RYSDE)]. The enzyme activity and overall enzyme productivity were respectively 16 U/ml and 0.19 U/ml h in the batch culture. The enzyme activity increased to 85 U/ml by feeding of partially hydrolyzed raw yam starch. When a mixture containing partially hydrolyzed raw yam starch and peptone was fed by a pH-stat strategy, the enzyme activity reached 366 U/ml, 23-fold of that obtained in the batch culture, and the overall productivity reached 3.4 U/ml h, which was 18-fold of that in the batch culture.     

Alteration of the properties of Aspergillus sp. K-27 glucoamylase on limited proteolysis with subtilisin

An active derivative (mol. wt. 48,000) of Aspergillus sp. K-27 glucoamylase (mol. wt. 76,000) was obtained by limited proteolysis with subtilisin. The amino acid sequences of native and modified enzymes at the N-termini were Ala-Gly-Gly-Thr-Leu-Asp and Ala-Val-Leu, respectively. The proteolysis greatly decreased the affinity of the enzyme for amylopectin and glycogen, but not for oligosaccharides. It also reduced the ability of the enzyme to degrade raw starch, abolished the ability of the enzyme to adsorb onto starch granules, and eliminated the synergistic action of the enzyme in the hydrolysis of starch granules with alpha-amylase. These findings imply that the enzyme has a specific affinity site for polysaccharide substrates besides the catalytic site, i.e., a starch-binding site, and that the former is removed by proteolysis. The extent of the reduction in the activity for raw starches caused by the modification varied with the starch source, as the modified enzyme digested raw potato starch better than either raw corn or sweet potato starches. A new method for evaluation of the raw starch-digesting activity of glucoamylase is described.     

Purification and characterization of a thermostable raw starch digesting amylase from a Streptomyces sp. isolated in a milling factory

A raw starch utilizing microbe was isolated from mud in a milling factory. The 16S ribosomal DNA (rDNA) sequencing and morphological properties of the strain indicated that it belongs to the genus Streptomyces. A strongly raw starch digesting amylase was purified from the culture supernatant of the strain by chromatographic procedures. The specific activity of the enzyme was 11.7 U/mg, molecular mass 47 kDa, optimum pH 6.0, and optimum temperature 50 to 60 degrees C. The enzyme showed sufficient activity even at 70 degrees C. It was activated by calcium, cobaltous, and magnesium ions, and inhibited by copper, nickel, zinc, and ferrous ions. It formed maltose mainly from raw and gelatinized starch, and glycogen. No products were formed from glucose, maltose, maltotriose, pullulan, or cyclodextrins (CDs). The enzyme digested raw wheat, rice, and waxy rice starch rapidly, and raw corn, waxy corn, sweet potato, tapioca, and potato starch normally.     

Behaviour of Endomycopsis fibuligera glucoamylase towards raw starch

An extracellular glucoamylase [exo-1,4-α-d-glucosidase, 1,4-α-d-glucan glucohydrolase, EC 3.2.1.3] of Endomycopsis fibuligera has been purified and some of its properties studied. It had a very high debranching activity (0.63). The enzyme was completely adsorbed onto raw starch at all the pH values tested (pH 2.0–7.6). Amylase inhibitor from Streptomyces sp. did not prevent the adsorption of glucoamylase onto raw starch although the enzyme did not digest raw starch in the presence of amylase inhibitor. Sodium borate (0.1 m) eluted only 35% of the adsorbed enzyme from raw starch. The optimum pH for raw starch digestion was 4.5 whereas that of boiled soluble starch hydrolysis was 5.5. Waxy starches were more easily digested than non-waxy starches, and root starches were slowly digested by this enzyme.     

The raw starch-degrading alkaline amylase ofBacillus sp IMD 370

The amylase ofBacillus sp IMD 370 is the first report of an alkaline amylase with the ability to digest raw starch. The amylase could degrade raw corn and rice starches more effectively than raw potato starch. It showed no adsorb-ability to any type of raw starch at any pH value tested. The enzyme digested raw corn starch to glucose, maltose, maltotriose and maltotetraose. The maximum pH for raw starch hydrolysis was pH 8.0 compared to pH 10.0 for soluble starch hydrolysis. The metal chelator, ethylenediaminetetraacetic acid, strongly inhibited raw starch-digestion and its effect was reversed by the addition of divalent cations. Degradation of raw starch was stimulated six-fold in the presence of -cyclodextrin (17.5 mM).     

Adsorption-elution purification of chimeric <Emphasis Type="Italic">Bacillus stearothermophilus</Emphasis> leucine aminopeptidase II with raw-starch-binding activity

Summary A chimericBacillus stearothermophilus leucine aminopeptidase II (LAPsbd) has been constructed by introducing the raw-starch-binding domain of Bacillus sp. strain TS-23 α-amylase into the enzyme. LAPsbd was adsorbed onto raw starch and the adsorbed enzyme could be eluted from the adsorbent by soluble starch in 20 mM Tris–HCl buffer (pH 8.0). The adsorption of LAPsbd onto raw starch was affected by raw starch concentration, pH, and temperature, while the temperature and incubation time had no obvious effects on the elution of adsorbed enzyme. The molecular weight of purified enzyme was estimated to be 61 kDa. About 84% of LAPsbd in the cell free extract was recovered through one adsorption–elution cycle with a purification of 20-fold. The high quantity and purity of the recovered enzyme coupled with the easy performance make the adsorption–elution procedure suitable for industrial applications.     

Preferential and rapid degradation of raw rice starch by an α-amylase of glycoside hydrolase subfamily GH13_37

The α-amylase (AmyP) from a marine metagenomic library belongs to the recently classified glycoside hydrolase subfamily GH13_37. The degradation abilities of AmyP on a broad range of raw starch granules were examined at 40 °C and pH 7.5. It was found that AmyP is a raw starch-degrading enzyme, exhibiting a unique and remarkable ability to preferentially and very rapidly digest raw rice starch. The specific activity of raw rice starch was reached 118.5?±?0.6 Umg(-1), which was much higher than that of other raw starches. The final hydrolysis degrees were obtained in 4 h for 1 % raw rice starch and 1 h for 8 % concentration, indicating a very rapid speed of hydrolysis. The presence of a starch residue resistant was the main limiting factor for complete hydrolysis, although end product inhibition also existed, especially at high starch concentrations. AmyP randomly attacks unique or susceptible sites on raw rice starch granules, and releases glucose, maltose, and maltotriose as end products. This is the first biochemical characterization of the raw starch-degrading ability of an α-amylase of family GH13_37. The specific ability towards raw rice starch has never been described before, and this makes AmyP a promising candidate for use as a novel enzyme in rice starch processing.     

海洋环境来源的淀粉酶AmyP对生玉米 淀粉的降解特性

来自海洋宏基因组文库的 α-淀粉酶（AmyP）属于最新建立的糖苷水解酶亚家族GH13₃7。AmyP 是一个生淀粉降解酶,能有效降解玉米生淀粉。在最适反应条件 pH 7.5和 40 °C 下,生玉米淀粉的比活达到 39.6 ± 1.4 U/mg。酶解反应动力学显示 AmyP 可以非常快速的降解生玉米淀粉。对 1%的生玉米淀粉仅需要 30 min;4%和 8%的生玉米淀粉只需 3 h。DTT 可以显著提高 AmyP 对生玉米淀粉的降解活性,1% DTT 促使活性增加 1倍。根据电镜观察和产物分析,认为 AmyP 是以内腐蚀的模式降解生玉米淀粉颗粒,释放出葡萄糖、麦芽糖和麦芽三糖作为终产物。     

Mutagenesis of echinocandin B overproducing Aspergillus nidulans capable of using starch as main carbon source

Abstract

Echinocandin B, a kind of antimycotic with cyclic lipo-hexapeptides, was produced by fermentation with Aspergillus nidulans using fructose as main carbon source. The objective of this study was to screen a high-yield mutant capable of using cheap starch as main carbon source by atmospheric and room temperature plasma (ARTP) treatment in order to decrease the production cost of echinocandin B. A stable mutant A. nidulans ZJB19033, which can use starch as optimal carbon source instead of expensive fructose, was selected from two thousands isolates after several cycles of ARTP mutagenesis. To further increase the production of echinocandin B, the optimization of fermentation medium was performed by response surface methodology (RSM), employing Plackett-Burman design (PBD) followed by Box-Behnken design (BBD). The optimized fermentation medium provided the optimal yield of echinocandin B, 2425.9?±?43.8?mg/L, 1.3-fold compared to unoptimized medium. The results indicated that the mutant could achieve high echinocandin B production using cheap starch as main carbon source, and the cost of carbon sources in fermentation medium reduced dramatically by about 45%.     

Preparation of Triglyceride Hydroperoxides

Corticium rolfsii AHU 9627, which we isolated from a tomato stem, is one of the most promising producers of a raw starch saccharifying enzyme. The effects of the cultural conditions and medium components on the enzyme production were investigated. The enzyme production was improved by increasing both the concentrations of carbon sources and organic nutrients in the medium. Under the optimum cultural conditions, the enzyme activity of the culture supernatant against raw starch reached a maximum after 8-days incubation at 27°C and the activity reached 80 units per ml (when determined at 40°C and pH 4.0). The optimal pH and temperature for the enzyme reaction were 4.0 and 65°C, respectively. The saccharifying reaction was scarcely inhibited even with a high substrate concentration, and raw starch was rapidly hydrolyzed into glucose.     

Production and Characteristics of Raw-Potato-Starch-Digesting alpha-Amylase from Bacillus subtilis 65

A newly isolated bacterium, identified as Bacillus subtilis 65, was found to produce raw-starch-digesting alpha-amylase. The electrophoretically homogeneous preparation of enzyme (molecular weight, 68,000) digested and solubilized raw corn starch to glucose and maltose with small amounts of maltooligosaccharides ranging from maltotriose to maltoheptaose. This enzyme was different from other amylases and could digest raw potato starch almost as fast as it could corn starch, but it showed no adsorbability onto any kind of raw starch at any pH. The mixed preparation with Endomycopsis glucoamylase synergistically digested raw potato starch to glucose at 30 degrees C. The raw-potato-starch-digesting alpha-amylase showed strong digestibility to small substrates, which hydrolyzed maltotriose to maltose and glucose, and hydrolyzed p-nitrophenyl maltoside to p-nitrophenol and maltose, which is different from the capability of bacterial liquefying alpha-amylase.     

Production and Characteristics of Raw Starch-Digesting Glucoamylase O from a Protease-Negative, Glycosidase-Negative Aspergillus awamori var. kawachi Mutant

Production of a raw starch-digesting glucoamylase O (GA O) by protease-negative, glycosidase-negative mutant strain HF-15 of Aspergillus awamori var. kawachi was undertaken under submerged culture conditions. The purified GA O was electrophoretically homogeneous and similar to the parent glucoamylase I (GA I) in the hydrolysis curves toward gelatinized potato starch, raw starch, and glycogen and in its thermostability and pH stability, but it was different in molecular weight and carbohydrate content (250,000 and 24.3% for GA O, 90,000 and ca. 7% for GA I, respectively). The chitin-bound GA O hydrolyzed raw starch but the chitin-bound GA I failed to digest raw starch because chitin was adsorbed at the raw starch affinity site of the GA I molecule. The removal of the raw starch affinity site of GA O with subtilisin led to the formation of a modified GA O (molecular weight, 170,000), which hydrolyzed glycogen 100%, similar to GA O and GA I, and was adsorbed onto chitin and fungal cell wall but not onto raw starch, Avicel, or chitosan. The modified GA I (molecular weight, 83,000) derived by treatment with substilisin hydrolyzed glycogen up to only 80% and failed to be adsorbed onto any of the above polysaccharides. The N-bromosuccinimide-oxidized GA O lost its activity toward gelatinized and raw starches, but the abilities to be adsorbed onto raw starch and chitin were preserved. It was thus suggested that both the raw starch affinity site essential for raw starch digestion and the chitin-binding site specific for the binding with chitin in the cell wall could be different from the active site, located in the three respective positions in the GA O molecule.     

Analysis of the raw starch-binding domain by mutation of a glucoamylase from Aspergillus awamori var. kawachi expressed in Saccharomyces cerevisiae.

Carboxy-terminal deletions were introduced into the raw starch-binding domain (A-515 to R-615) encoded by the gene for glucoamylase I (GAI) from Aspergillus awamori var. kawachi. Genes coding for proteins designated GA596 (A-1 to E-596), GA570 (A-1 to A-570), and GA559 (A-1 to N-559) were constructed and resulted in truncated proteins. All of the mutant genes were expressed heterologously in Saccharomyces cerevisiae. GA596 adsorbed to raw starch and digested it. GA570 and GA559 did not adsorb to raw starch or to an alpha-cyclodextrin-Sepharose CL-4B gel under our experimental conditions. However, GA570 was able to digest raw starch, and the digestion of raw starch by GA570 was inhibited by beta-cyclodextrin. Residue Trp-562 of GAI, which was suggested previously to contribute to formation of an inclusion complex with raw starch, was replaced by Leu (GAW562L), Phe (GAW562F), and Gly (GAW562G). GAW562L and GAW562F adsorbed to raw starch and an alpha-cyclodextrin gel, but GAW562G did not. Although GAW562L digested raw starch to the same extent as wild-type GAI (designated GAY), GAW562F and GAW562G exhibited less ability to digest raw starch. On the basis of our results, it appears that the sequence around Trp-562, PL(W-562)YVTVTLPA, is the minimal sequence necessary for digestion of raw starch and that hydrophobic residue Trp-562 contributes to formation of an inclusion complex. The sequence near Trp-589, which has abundant hydrogen bond-forming residues and the charged amino acid residues needed for stable adsorption to raw starch, probably assists in the formation of the inclusion complex.     

A novel raw starch digesting alpha-amylase from a newly isolated Bacillus sp. YX-1: purification and characterization

This study reports the purification and characterization of a novel raw starch digesting alpha-amylase from a newly isolated Bacillus sp. YX-1. Maximum alpha-amylase activity (53 U mL(-1)) was obtained at 45 degrees C after 44 h of incubation. The enzyme was purified using ammonium sulfate precipitation, ion exchange and gel filtration chromatography, and showed a molecular weight of 56 kDa by SDS-PAGE. This enzyme exhibited maximum activity at pH 5.0, performed stability over a broad range of pH 4.5-11.0, and was optimally active at 40-50 degrees C. The enzyme preparation had a strong digesting ability towards various raw starches and efficiently hydrolyzed raw corn starch at a concentration of 20% and pH 5.0, which were normally used in the starch industries, in a period of 12h. By analyzing its partial amino acid sequences, the enzyme was proposed to be a novel alpha-amylase.     

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.