Tailoring Pullulanase PulAR from Anoxybacillus sp. AR-29 for Enhanced Catalytic Performance by A Structure-Guided Consensus Approach

Pullulanase is a well-known debranching enzyme that can specially hydrolyze α-1,6-glycosidic linkages in starch and oligosaccharides, however, it suffers from low stability and catalytic efficiency under industrial conditions. In the present study, four sites (A365, V401, H499, and T504) lining the catalytic pocket of Anoxybacillus sp. AR-29 pullulanase PulAR were selected for site-directed mutagenesis (SDM) by using a structure-guided consensus approach. Four beneficial mutants (PulAR-A365V, PulAR-V401C, PulAR-A365/V401C, PulAR-A365V/V401C/T504V, and PulAR-A365V/V401C/T504V/H499A) were created, which showed enhanced thermostability, pH stability, and catalytic efficiency. Among them, the quadruple mutant PulAR-A365V/V401C/T504V/H499A displayed 6.6- and 9.6-fold higher catalytic efficiency toward pullulan at 60 ℃, pH 5.0 and 6.0, respectively. In addition, its thermostabilities at 60 ℃ and 65 ℃ were improved by 2.6- and 3.1-fold, respectively, compared to those of the wild-type (WT). Meanwhile, its pH stabilities at pH 4.5 and 5.0 were 1.6- and 1.8-fold higher than those of WT, respectively. In summary, the catalytic performance of PulAR was significantly enhanced via rational engineering by a structure-guided consensus approach. The resultant quadruple mutant PulAR-A365V/V401C/T504V/H499A demonstrated potential applications in the starch industry.

Development of production of natural and modified starches in the starch syrup industry

Анализ работы крахмалопаточной отрасли по производству нативных и модифицированных крахмалов за период 2010-2020 гг. выявил положительные тенденции, характеризующиеся умеренными темпами роста выпуска продукции. Увеличение объемов производства нативных и модифицированных крахмалов, расширение их ассортимента - актуальная задача для предприятий крахмалопаточной отрасли. В статье приведен ассортимент нативных и модифицированных крахмалов, поставляемых на российский рынок. Большой интерес для отраслей пищевой промышленности и народного хозяйства в целом представляют эфиры крахмала холодного и горячего набухания, доля которых в общем объеме импорта модифицированных крахмалов составляет около 70 %. Приведен перечень высокотехнологичных крахмалопаточных предприятий, обеспечивающих высокое качество и конкурентоспособность производимой продукции. Дана динамика производства нативных крахмалов и потребления модифицированных крахмалов в РФ в 2010-2020 гг. За последние 10 лет объемы производства модифицированных крахмалов в крахмалопаточной отрасли увеличились в 2,3 раза. Приведены современные методы получения и области применения модифицированных крахмалов для пищевого назначения. Предложены механизмы устойчивого развития крахмалопаточных предприятий в современных условиях. An analysis of the work of the starch syrup industry in the production of natural and modified starches for the period 2010-2020 revealed positive trends characterized by a moderate growth rate in production. Increasing the natural and modified starches production volume, expanding the range is an urgent task for enterprises of the starch industry. The article provides an assortment of natural and modified starches supplied to the Russian market. Cold and hot swell starch esters are of great interest for the food industry and the national economy as a whole, which account for about 70 % of the total imports of modified starches. The list of high-tech starch processing enterprises ensuring high quality and competitiveness of the produced products is given. The dynamics of production of natural starches and the dynamics of production and consumption of modified starches in the Russian Federation in the period 2010-2020 are given. Over the past 10 years, the production of modified starches in the starch industry has increased 2.3 times. Modern methods of producing and application of modified starches for food purposes are presented. Mechanisms for sustainable development of starch-syrup enterprises in modern conditions are proposed.

Bioconversion of Industrial Cassava Solid Waste (Onggok) to Bioethanol Using a Saccharification and Fermentation process

Cassava solid waste (Onggok) is a by-product of the starch industry containing a lot of fiber, especially cellulose and hemicellulose. It has the potential to be converted to bioethanol. This work aimed to evaluate the effect of feedstocks ratio for the optimal bioethanol production via enzymatic and acidic hydrolysis process in a batch fermentation process. The effect of alpha-amylase and glucoamylase activities was studied. The sulfuric acid concentrations in the hydrolysis process in converting cassava into reducing sugar were also investigated. The reducing sugar was then fermented to produce ethanol. Enzymatic and chemical hydrolysis was carried out with the ratio of onggok(g)/water(L), 50/1, 75/1, and 100/1 (w/v). In the enzymatic hydrolysis, 22.5, 45, and 67.5 KNU (Kilo Novo alpha-amylase Unit) for liquefaction; and 65, 130, and 195 GAU (Glucoamylase Unit) for saccharification, respectively of enzymes were applied. The liquefaction was carried out at 90-100⁰C for 2 hours. The saccharification was executed at 65 ⁰C for 4 hours. Meanwhile, the acidic hydrolysis operating condition was at 90-100 ⁰C for 3 hours. The fermentation was performed at pH 4.5 for 3 days. Fourier Transform Infra-Red (FTIR) analysis was conducted to evaluate the hydrolysis process. The highest ethanol was yielded in the fermentation at 8.89% with the ratio of onggok to water 100:1, 67.5 KNU of alpha-amylase, and 195 GAU of glucoamylase. Ethanol was further purified utilizing fractional distillation. The final ethanol concentration was at 93-94%.

Deep processing of starch-containing raw materials: current state and prospects for sustainable development

Современные требования инновационной экономики к перерабатывающему сектору сельскохозяйственного сырья должны ориентироваться на безотходные ресурсосберегающие технологии переработки, экологизации производства и природосбережения, в этом и заключается философия технологии глубокой переработки. Только заложив в основу хозяйственной деятельности эти принципы работы, можно обеспечить полное использование всех имеющихся компонентов сельскохозяйственного сырья при минимальном получении вторичных ресурсов и отходов производства и обеспечить устойчивое развитие промышленности. Глубокая переработка зерна кукурузы и пшеницы в России является наиболее перспективным направлением, для этого создана развитая сырьевая база и построены современные заводы по переработке этих видов сырья с производством значительного ассортимента продукции. Разработка и внедрение в промышленность технологий глубокой переработки на основе диверсификации производства с получением широкой номенклатуры продукции является ключевым приоритетом развития крахмалопаточной промышленности России. Необходимость обеспечения развития глубокой переработки зерна обусловлена рядом факторов - это связано с удовлетворением внутреннего спроса на данную продукцию, снижением импортной зависимости от продуктов переработки зерна с высокой добавленной стоимостью, возможностью для выхода на международные рынки в рамках федерального проекта «Экспорт продукции АПК». Последнее десятилетие для крахмалопаточной промышленности было отмечено ростом производственных мощностей, модернизацией действующих предприятий, концентрацией производства, что расширило диверсификацию экономики промышленности и обеспечило хорошую динамику выработки основных видов продукции, повысило ее конкурентоспособность. Комплексное решение стоящих перед промышленностью задач и вывод ее на новый технологический уровень возможны только при разработке целевой программы развития, которая свяжет использование всех видов ресурсов на основе современных достижений научно-технического прогресса и новых форм организации производства. The modern requirements of the innovative economy for the processing sector of agricultural raw materials should focus on waste-free resource-saving technologies for processing, greening production and environmental conservation, this is the philosophy of deep processing technology. Only by laying the foundation for economic activity can these principles of work ensure the full use of all available components of agricultural raw materials with a minimum of secondary resources and production waste and ensure the sustainable development of industry. The deep processing of corn and wheat grains in Russia is the most promising area, for this purpose, a developed raw material base has been created and modern plants for the processing of these raw materials have been built with the production of a significant range of products. The development and introduction into the industry of deep processing technologies based on diversification of production to obtain a wide range of products is a key priority for the development of the Russian starch industry. The need to ensure the development of deep grain processing is due to a number of factors - this is due to the satisfaction of domestic demand for these products, a decrease in import dependence on high-value-added grain processing products, and the possibility of entering international markets under the federal project «Export of agro-industrial products». The last decade for the starch industry was marked by an increase in production capacities, modernization of existing enterprises, a concentration of production, which expanded the diversification of the economy of industry and ensured good dynamics of production of basic types of products, increased its competitiveness. It is possible to comprehensively solve the problems facing industry and bring it to a new technological structure only when developing a targeted development program that will link the use of all types of resources based on modern achievements in scientific and technological progress and new forms of production organization.

Improvement of thermostability and catalytic efficiency of glucoamylase from Talaromyces leycettanus JCM12802 via site-directed mutagenesis to enhance industrial saccharification applications

Background Glucoamylase is an important industrial enzyme in the saccharification of starch into glucose. However, its poor thermostability and low catalytic efficiency limit its industrial saccharification applications. Therefore, improving these properties of glucoamylase is of great significance for saccharification in the starch industry. Results In this study, a novel glucoamylase-encoding gene TlGa15B from the thermophilic fungus Talaromyces leycettanus JCM12802 was cloned and expressed in Pichia pastoris. The optimal temperature and pH of recombinant TlGa15B were 65 ℃ and 4.5, respectively. TlGa15B exhibited excellent thermostability at 60 ℃. To further improve thermostability without losing catalytic efficiency, TlGa15B-GA1 and TlGa15B-GA2 were designed by introducing disulfide bonds and optimizing residual charge–charge interactions in a region distant from the catalytic center. Compared with TlGa15B, mutants showed improved optimal temperature, melting temperature, specific activity, and catalytic efficiency. The mechanism underlying these improvements was elucidated through molecular dynamics simulation and dynamics cross-correlation matrices analysis. Besides, the performance of TlGa15B-GA2 was the same as that of the commercial glucoamylase during saccharification. Conclusions We provide an effective strategy to simultaneously improve both thermostability and catalytic efficiency of glucoamylase. The excellent thermostability and high catalytic efficiency of TlGa15B-GA2 make it a good candidate for industrial saccharification applications.

Potato Juice, a Starch Industry Waste, as a Cost-Effective Medium for the Biosynthesis of Bacterial Cellulose

In this work, we verified the possibility of valorizing a major waste product of the potato starch industry, potato tuber juice (PJ). We obtained a cost-effective, ecological-friendly microbiological medium that yielded bacterial cellulose (BC) with properties equivalent to those from conventional commercial Hestrin–Schramm medium. The BC yield from the PJ medium (>4 g/L) was comparable, despite the lack of any pre-treatment. Likewise, the macro- and microstructure, physicochemical parameters, and chemical composition showed no significant differences between PJ and control BC. Importantly, the BC obtained from PJ was not cytotoxic against fibroblast cell line L929 in vitro and did not contain any hard-to-remove impurities. The PJ-BC soaked with antiseptic exerted a similar antimicrobial effect against Staphylococcus aureus and Pseudomonas aeruginosa as to BC obtained in the conventional medium and supplemented with antiseptic. These are very important aspects from an application standpoint, particularly in biomedicine. Therefore, we conclude that using PJ for BC biosynthesis is a path toward significant valorization of an environmentally problematic waste product of the starch industry, but also toward a significant drop in BC production costs, enabling wider application of this biopolymer in biomedicine.

Potato juice, a starch industry waste, as a cost-effective medium for the biosynthesis of bacterial cellulose

The unique properties of bacterial cellulose (BC) make it of great interest for numerous branches of industry. Nevertheless, the high cost of the dedicated, microbiological medium used for BC production significantly hinders possibility of widespread use. Searching for an alternative, we turned our attention to potato tuber juice (PJ), a major waste product of the potato starch industry. We verified the possibility of using PJ as a cost-effective, ecological-friendly medium that yielded BC with properties equivalent to those from conventional commercial Hestrin-Schramm medium. The BC yield from PJ medium (>4 g/L) was comparable, despite the lack of any pre-treatment. Likewise, the macro- and microstructure, physicochemical parameters, and chemical composition showed no significant differences between PJ and control BC. Importantly, BC obtained from PJ was not cytotoxic against fibroblast cell line L929 in vitro and did not contain any hard-to-remove impurities. These are very important aspects from an application standpoint, particularly in biomedicine. Therefore, we conclude that using PJ for BC biosynthesis is a path towards significant valorization of an environmentally problematic waste product of the starch industry and can help ultimately lower BC production costs.