Genetic engineering of cyclomaltodextrin glucanotransferases

Studies of cyclic oligosaccharides from six, seven and eight glucose residues, designated as alpha-, beta- and gamma-cyclodextrins, respectively, and everything related to them have been going on for 130 years. In this review, the history of the study of these molecules is briefly considered. The interest in cyclodextrins is caused by their ability to form inclusion complexes with a number of organic and inorganic compounds, radically changing some of their properties. This is widely used in the pharmaceutical, cosmetic and food industries, and beta-cyclodextrin is even registered as a food additive E459. Cyclodextrins are obtained from starch under the action of cyclodextringlucanotransferase (CGTase) enzymes, a characteristic feature of which is their non-strict specificity in relation to the types of oligosaccharides produced. The main producers of these enzymes are a group of bacteria of the order Bacillales, which unites several families (Paenibacillaceae, Bacillaceae, Thermoactynomicetaceae, etc.), but in last years CGTases have been found in a wide range of bacteria and archaea. The genetic engineering of CGTases began in the middle of 1980s, after the CGTase gene from Paenibacillus macerans (formerly Bacillus macerans) was cloned and sequenced for the first time, and during this period rather noticeable progress was made in understanding the organization and functioning of these enzymes, including using X-ray diffraction analysis. With the help of site-directed mutagenesis, error-prone PCR, as well as by creating chimeric forms of these enzymes, certain successes have been achieved in recent decades in changing (improving) the specificity of their action. Suitable leader peptides are used to increase the synthesis and secretion of genetically engineered CGTases, and various heterologous producers are also proposed, including the bacteria Escherichia coli, B.subtilis, Lactococcus lactis and the methylotrophic yeast Koagataella phaffii.

THE EFFECT OF CYCLODEXTRIN ON THE DESTRUCTION OF PHENOL BY BACTERIA PSEUDOMONAS STUTZERI AND PAENIBACILLUS MACERANS IN THE AQUATIC ENVIRONMENT

Cyclodextrins (CD) by their chemical nature are typical "host molecules" that can encapsulate a wide range of molecules, one or two benzene rings, including a phenolic ring. CDs can affect the bioavailability of contaminants during biodegradation, as well as the toxicity of the contaminant to microorganisms due to their ability to form inclusion complexes with organic compounds. In this work, the phenol destructor strains Pseudomonas stutzeri IB-I6C and Paenibacillus macerans IB-I4 were used, which have different growth characteristics in relation to the tested substrates: phenol (200 µg/ml) and CD (2 mg/ml). It was shown that when cultivating strains on a nutrient medium containing phenol+cyclodextrin, the degradation potential of bacteria in relation to phenol significantly increases. The amount of residual phenol in the presence of CD for P. stutzeri IB-I6C culture decreased by 8% by the end of cultivation, and for P. macerans IB-I4 this value was 10%. In the presence of CD in the medium, the time to achieve the maximum possible destruction of phenol was almost halved for both destructor strains. The rate of phenol consumption by pseudomonas IB-I6C in the presence of CD increased by 1,36 times, and utilization by the P. macerans strain IB-I4 was 1,31 times faster. The positive effect of CD in the destruction of phenol was found even in the case when CD is poorly utilized by the microorganism, as is observed in the strain P. stutzeri IB-I6C.

Expression of Gene for β-glucanase from Paenibacillus jamilae Bg1 in Pichia pastoris and Characteristics of Recombinant Enzyme

The isolation, heterologous expression and characterization of a new thermostable β-glucanase from Paenibacillus jamilae is described. The bgl26 gene from the P. jamilae Bg1 VKPM B-13093 strain consisting of 714 nucleotides encodes endo-1,3-1,4-β-glucanase (EC 3.2.1.73) containing 213 amino acids and 24 residues of the putative signal peptide in N-end area. The nucleotide sequence of the bgl26 gene and the amino acid sequence of the mature Bgl26 protein have the greatest homology with the sequence of the Paenibacillus macerans endo-l,3-l,4-β-glucanase (82 and 88%, respectively). A fragment of the gene encoding the mature protein was expressed in Pichia pastoris. Purified recombinant enzyme Bgl26 was active towards barley β-glucan. The optimal pH for the enzyme to work was 7,0, and the optimum temperature range was 40-45 °C. The specific activity of β-glucanase was at the level of 6650 U/mg of protein, Km and Vmax were equal to 6.4 ± 0.3 mg/mL and 9450.1 ± 471.2 umol/(min-mg), respectively. The recombinant protein Bgl26 was characterized by high pH and thermal stability, as well as resistance to digestive enzymes. It is also shown that Co2+ ions have a positive effect on the activity of the enzyme. β-glucanase, β-glucan, Paenibacillus jamilae, Pichia pastoris The work was financially supported by the Ministry of Science and Higher Education of the Russian Federation (Unique Project Identifier RFMEFI60717X0179) and was carried out using the Multipurpose Scientific Installation of National Bio-Resource Center «All-Russian Collection of Industrial Microorganisms», NRC «Kurchatov Institute» - GOSNIIGENETIKA.

Reclamation of Marine Chitinous Materials for Chitosanase Production via Microbial Conversion by Paenibacillus macerans

Chitinous materials from marine byproducts elicit great interest among biotechnologists for their potential biomedical or agricultural applications. In this study, four kinds of marine chitinous materials (squid pens, shrimp heads, demineralized shrimp shells, and demineralized crab shells) were used to screen the best source for producing chitosanase by Paenibacillus macerans TKU029. Among them, the chitosanase activity was found to be highest in the culture using the medium containing squid pens as the sole carbon/nitrogen (C/N) source. A chitosanase which showed molecular weights at 63 kDa was isolated from P. macerans cultured on a squid pens medium. The purified TKU029 chitosanase exhibited optimum activity at 60 °C and pH 7, and was stable at temperatures under 50 °C and pH 3-8. An analysis by MALDI-TOF MS revealed that the chitosan oligosaccharides (COS) obtained from the hydrolysis of water-soluble chitosan by TKU029 crude enzyme showed various degrees of polymerization (DP), varying from 3–6. The obtained COS enhanced the growth of four lactic acid bacteria strains but exhibited no effect on the growth of E. coli. By specialized growth enhancing effects, the COS produced from hydrolyzing water soluble chitosan with TKU029 chitinolytic enzymes could have potential for use in medicine or nutraceuticals.