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Starch is an important food ingredient and a substrate for the production of many industrial products. Biological and industrial processes involve hydrolysis of raw starch, such as digestion by humans and animals, starch metabolism in plants, and industrial starch conversion for obtaining glucose, fructose and maltose syrup or bioethanol. Raw starch degrading α-amylases (RSDA) can directly degrade raw starch below the gelatinization temperature of starch. Knowledge of the structures and properties of starch and RSDA has increased significantly in recent years. Understanding the relationships between structural peculiarities and properties of RSDA is a prerequisite for efficient application in different aspects of human benefit from health to the industry. This review summarizes recent advances on RSDA research with emphasizes on representatives of glycoside hydrolase family GH13. Definite understanding of raw starch digesting ability is yet to come with accumulating structural and functional studies of RSDA.

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Captive breeding has been suggested as a method of conservation for many vertebrates, and is increasingly being proposed as a strategy for invertebrates. In this study, the growth, development and fertility of adults of the vulnerable cerambycid Morimus funereus reared in captivity are examined. Two oviposition cycles; from May to September and from January to March were studied and larvae from wild adults and from the progeny of captive adults (second generation larvae) were examined. Five to 12 instars were observed during larval development. Larval development was completed in 218 days (average) for the progeny of wild adults with an average mortality rate of 10.3% and in 226 days (average) for larvae from captive adults with mortality rate of 34.9%. First generation larval body weights were disparate during development, while second generation larvae had similar weights with no significant differences. In this study we have tested the potential of captive breaded M. funereus larvae as a model for investigation of digestive enzymes. Amylase from the midgut of larvae reared under laboratory conditions showed twofold higher specific activities with a decreased number of isoforms expressed, as compared to the enzyme from field-collected larvae. Captive breeding of M. funereus can be used in the future as a part of an effective conservation strategy for this rare insect species.

Bacillus paralicheniformis (BliAmy), belonging to GH13_5 subfamily of glycoside hydrolases, was proven to be a highly efficient raw starch digesting enzyme. The ability of some α-amylases to hydrolyze raw starch is related to the existence of surface binding sites (SBSs) for polysaccharides that can be distant from the active site. Crystallographic studies performed on BliAmy in the apo form and of enzyme bound with different oligosaccharides and oligosaccharide precursors revealed binding of these ligands to one SBS with two amino acids F257 and Y358 mainly involved in complex formation. The role of this SBS in starch binding and degradation was probed by designing enzyme variants mutated in this region (F257A and Y358A). Kinetic studies with different substrates show that starch binding through the SBS is disrupted in the mutants and that F257 and Y358 contributed cumulatively to binding and hydrolysis. Mutation of both sites (F257A/Y358A) resulted in a 5-fold lower efficacy with raw starch as substrate and at least 5.5-fold weaker binding compared to the wild type BliAmy, suggesting that the ability of BliAmy to hydrolyze raw starch with high efficiency is related to the level of its adsorption onto starch granules.

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Please cite this article as: Nikola Lončar, Nataša Božić, Zoran Vujčić, Expression and characterization of a thermostable organic solevent-tolerant laccase from Bacillus licheniformis ATCC 9945a, Journal of Molecular Catalysis B: Enzymatic http://dx.Abstract Highlights  Bacillus licheniformis 9945a laccase is overexpressed in E. coli with yield 50 mg/L.  Temperature optimum of laccase is 90⁰C and pH optimum is 7.0. Enzyme is thermostable with a melting temperature of 79⁰C at pH 7.0. Presence of organic solvents reduces melting temperature but activity remains impaired. Lignin model compounds are dimerized after one electron oxidation of phenolic group. AbstractBacterial laccases have proven advantages over fungal and plant counterparts in terms of wider pH optimum, higher stability and broader biocatalytic scope. In this work, Bacillus licheniformis ATCC 9945a laccase is produced heterologously in Escherichia coli. Produced laccase exhibits remarkably high temperature optimum at 90⁰C and possess significant thermostability and resistance to inactivation by organic solvents. Laccase has an apparent melting temperature of 79⁰C at pH 7.0 and above 70⁰C in range of pH 5.0-8.0, while having half-life of 50 min at 70⁰C. Presence of 10% organic solvents such as acetonitrile, dimethylformamide, dimethylsulfoxide or methanol reduces melting temperature to 45-52ᴼC but activity remains practically unimpaired. With 50% of acetonitrile and methanol laccase retained 3 ~40% of initial activity. EDTA and 300 mM sodium-chloride have positive effect on activity.Enzyme is active on syringaldazine, ABTS, phenols, amines, naphthol, lignin and lignin model compounds and mediates C-C bond formation via oxidative coupling after one electron oxidation of phenolic group. Successful polymerization of 2-naphthol was achieved with 77% conversion of 250 mg/L 2-naphtol in only 15 min which may further expand substrate scope of this enzyme towards polymer production and/or xenobiotics removal for environmental applications.