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The heterologous expression and characteristics of a new xylanase from Pyromyces finnis have been described. The endo-l,4-β-xylanase XylP (EC 3.2.1.8) consists of 223 amino acids and 19 residues of a putative signal peptide in the N-terminal region. The amino acid sequence of the mature protein has the greatest homology with the sequence of the native catalytic N-terminal domain of Neocallimastix patriciarum endo-l,4-β-xylanase (84%). A synthetic nucleotide sequence encoding a mature XylP protein was expressed in Pichia pastoris. The purified recombinant enzyme showed activity with birch xylan and arabinoxylan. When using birch xylan as a substrate, the optimum pH for the enzyme was 5.0, and the optimum temperature was 50 °C. The specific activity of the xylanase was 4700 U/mg protein, and Km and Vmax were equal to 0.51 mg/mL and 7395.3 umol/(min∙mg), respectively. The recombinant XylP protein showed moderate thermal stability and high pH stability, resistance to digestive enzymes and protein inhibitors of grain xylanases. It was also shown that the Mg2+, Co2+ and Li+ ions have a positive effect on the enzyme activity. xylanase, xylan, feed enzyme, Pichia pastoris, Pyromyces finnis The work was performed with the financial support of the Ministry of Education and Science of Russia (Unique Project Identifier RFMEFI60717X0180) using the Unique Scientific Installation -National Bioresource Center «All-Russian Collection of Industrial Microorganisms», NRC «Kurchatov Institute» - GOSNIIGENETIKA

HepI to maltose-binding protein (MBP) and succeeded in its high-yield production and 66 easy purification. As a result, approximately 90% of protein was in soluble heparinase active 67 form. And in this case protein had specific activity of 88.3 units / mg of protein (reducing 68 specific activity by 25%) (Chen et al., 2005(Chen et al., , 2007. 69Nevertheless, even in the form fusion of maltose binding protein with heparinase I enzyme 70 shows low thermostability and specific activity. 71The C297S mutation had been introduced in the gene encoding heparinase I, in order to increase 72 the thermostability of the enzyme MBP-HepI, as well as the reaction conditions were chosen the 73 degradation of heparin. In the course of this work showed a significant increase in the 74 thermostability and residual enzymatic activity after prolonged storage (Chen et al.,2011(Chen et al., , 2013. 75Our group is involved in work the cost-effective and industry-applicable catalysis with 76 heparinase I through improving its productivity and activity. The purpose of our work was 77 increasing the specific activity of the enzyme, based on the data structure of the active site and 78 mechanism of action of heparinase I. 79Amino acid residues cysteine -135 and histidine-203 of heparinase I play a major role in the 80 cleavage of heparin. Thus, during the initiation of the reaction, the sulfur of the thiol group on 81 the cysteine-135 is formed a negative charge, then the thiolate anion performs nucleophilic 82 attack heparin, during which are formed the breaking of bonds in the oligomers and form 83 mukopolisaaharides. It should be noted that increasing the specific activity can be realized by 84 improving in enzyme stability in the form of the cysteine-135 thiolate anion (Sasisekharan, 85 1991). 86Our task was a search effective areas in sequence heparinase I and the introduction of mutations 87 that could lead to the stabilization of the negative charge on the thiol group of cysteine -135 in 88 order to increase the specific activity of enzyme. 89As a result was obtained the mutant enzyme heparinase I with a significantly increased specific 90 activity (approximately 25%). 91 Materials and methods 92 Materials, bacterial strains, plasmids and media 93 Heparin sodium salt (150 I.U./mg) was purchased from ApplliChem. Heparinase I 94 from Flavobacterium heparinum (≥200 U/mg) was purchased from Sigma-Aldrich. 95 F. heparinum (ATCC 13125) was purchased from the Institute of Applied Microorganisms 96 (IAM) of the University of Tokyo, Japan. E. coli Tuner DE3 was purchased from VKPM 97 (Russia). This strain was used for studying influence introduced mutations in a gene HepI on 98 activity of the enzyme. Plasmid pET22b(+) preserved in our laboratory was used.99

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The expression potential of various strains from the Collection of the National Bio-Resource Center (BRC VKPM) collection belonging to the species Komagataella kurtzmanii, K. phaffii, K. mondaviorum has been assessed by the level of production of the heterologous enzyme Citrobacter freundii phytase. Heterologous expression in the K. mondaviorum strains was observed for the first time. The strains of K. phaffii Y-4288, K. mondaviorum Y-4331 and K. phaffii Y-4287 were identified with a high level production of the heterologous enzyme, a high growth rate, the ability to accumulate a large amount of biomass and moderate thermotolerance. It was shown that the average productivity of the transformants based on K. phaffii Y-4288, K. mondaviorum Y-4331, and K. phaffii Y-4287 strains exceeds that of the commercial industrial recipient strain K. phaffii GS115 Y-2837 by more than 3, 5 and 6 times, respectively. The K. phaffii Y-4287 and K. mondaviorum Y-4331 strains exhibited moderate thermotolerance and the ability to accumulate a heterologous product at 37° C. The high expression potential of the identified strains opens up the possibility of creating recipient strains on their basis for high-level production of heterologous proteins. Key words: Komagataella, Pichia pastoris, thermotolerance, expression of heterologous proteins Funding - This work was supported by the Ministry of Science and Higher Education of the Russian Federation (grant no. 075-15-2019-1658 dated October 31, 2019) and was carried out using the resources of the Unique Scientific Facility of the "All-Russian Collection of Industrial Microorganisms" National Bio-Resource Center, NRC «Kurchatov Institute»---GOSNIIGENETIKA.